Induction of Nitrate Transport in Maize Roots, and Kinetics of Influx, Measured with Nitrogen-13

Abstract

Unlike phosphate or potassium transport, uptake of nitrate by roots is induced, in part, by contact with the substrate ion. Plasmalemma influx of (13)N-labeled nitrate in maize roots was studied in relation to induction of the uptake system, and the influence of short-term N starvation. Maize (Zea mays) roots not previously exposed to nitrate had a constitutive transport system (state 1), but influx increased 250% during six hours of contact with 100 micromolar nitrate, by which time the transport mechanism appeared to be fully synthesized (state 2). A three-day period of N starvation prior to induction and measurement of nitrate influx resulted in a greater capacity to transport nitrate than in unstarved controls, but this was fully expressed only if roots were kept in contact with nitrate for the six hours needed for full induction (state 2E). A kinetic analysis indicated a 160% increase in maximum influx in N-starved, induced roots with a small decrease in K(m). The inducible component to nitrate influx was induced only by contact with nitrate. Full expression of the nitrate inducible transport system was dependent upon mRNA synthesis. An inhibitor of cytoplasmic protein synthesis (cycloheximide) eliminated the formation of the transport system while inhibition by chloramphenicol of mitochondrial- or plastid-coded protein synthesis had no effect. Poisoning of membrane-bound proteins effectively disabled both the constitutive and induced transport systems.

Full text

Plant
Physiol.
(1990)
93,
642-647
0032-0889/90/93/0642/06/$01
.00/0
Received
for
publication
October
23,
1989
and
in
revised
form
January
31,
1990
Induction
of
Nitrate
Transport
in
Maize
Roots,
and
Kinetics
of
Influx,
Measured
with
Nitrogen-131
David
J.
Hole*,
Ali
M.
Emran,
Youhanna
Fares,
and
Malcolm
C.
Drew
Department
of
Horticultural
Sciences,
Texas
A&M
University,
College
Station,
Texas
77843-2133
(D.J.H.,
M.C.D.),
Positron
Diagnostic
and
Research
Center,
University
of
Texas
Health
Science
Center
at
Houston,
Texas
77030
(A.M.E.),
and
Biosystec
Inc.,
Neal
Pickett
Dr.,
College
Station,
Texas
77840
(Y.F.)
ABSTRACT
Unlike
phosphate
or
potassium
transport,
uptake
of
nitrate
by
roots
is
induced,
in
part,
by
contact
with
the
substrate
ion.
Plasmalemma
influx
of
13N-labeled
nitrate
in
maize
roots
was
studied
in
relation
to
induction
of
the
uptake
system,
and
the
influence
of
short-term
N
starvation.
Maize
(Zea
mays)
roots
not
previously
exposed
to
nitrate
had
a
constitutive
transport
system
(state
1),
but
influx
increased
250%
during
six
hours
of
contact
with
100
micromolar
nitrate,
by
which
time
the
transport
mecha-
nism
appeared
to
be
fully
synthesized
(state
2).
A
three-day
period
of
N
starvation
prior
to
induction
and
measurement
of
nitrate
influx
resulted
in
a
greater
capacity
to
transport
nitrate
than
in
unstarved
controls,
but
this
was
fully
expressed
only
if
roots
were
kept
in
contact
with
nitrate
for
the
six
hours
needed
for
full
induction
(state
2E).
A
kinetic
analysis
indicated
a
160%
increase
in
maximum
influx
in
N-starved,
induced
roots
with
a
small
decrease
in
Km.
The
inducible
component
to
nitrate
influx
was
induced
only
by
contact
with
nitrate.
Full
expression
of
the
nitrate
inducible
transport
system
was
dependent
upon
mRNA
synthesis.
An
inhibitor
of
cytoplasmic
protein
synthesis
(cyclo-
heximide)
eliminated
the
formation
of
the
transport
system
while
inhibition
by
chloramphenicol
of
mitochondrial-
or
plastid-coded
protein
synthesis
had
no
effect.
Poisoning
of
membrane-bound
proteins
effectively
disabled
both
the
constitutive
and
induced
transport
systems.
Absorption
of
nitrate
by
roots
provides
the
predominant
source
of
N
for
the
growth
and
yield
of
most
crop
species,
yet
the
internal
factors
regulating
its
uptake
from
the
soil
solution,
and
the
initial
stages
in
its
subsequent
metabolism
are
poorly
understood
(23).
Previous
studies
with
barley
have
shown
that
NO3-
influx
(14,
19)
as
well
as
net
uptake
(15)
are
stimulated
when
NO3-
is
supplied
following
a
period
of
NO3-
starvation
or
temporary
deprivation.
A
similar
stimulation
of
ion
trans-
port
following
temporary
deprivation
occurs
with
phosphate
(6,
12),
sulfate
(3),
and
potassium
(6,
8),
the
effect
being
specific
to
the
ion
previously
in
short
supply
(12).
Because
the
effect
is
not
a
generalized
one
on
salt
transport
by
roots,
it
implies
a
specific
modification
to
the
ion
transport
mecha-
nism
itself.
However,
unlike
all
other
ions
so
far
studied,
the
'
Research
supported
by
Texas
Agricultural
Experimental
Station
Project
H-6850,
and
by
a
grant
from
Texas
A&M
University
to
M.C.D.
Texas
Agricultural
Experiment
Station
Technical
Article
No.
25044.
constitutive
ability
to
transport
NO3-
is
poorly
expressed:
the
full
ability
to
transport
N03-
requires
induction
by
contact
with
the
substrate
ion,
NO3-
(14,
19).
Like
barley,
the
roots
of
maize
are
known
to
require
pre-
vious
contact
with
NO3-
for
full
induction
of
NO3-
transport
(1
1,
16,
20),
with
loss
of
transport
ability
during
periods
of
NO3-
deprivation
in
excess
of
24
to
48
h
(20).
Induction
of
N03-
transport
has
been
closely
associated
with
a
distinct
group
of
newly
synthesized
polypeptides
in
the
plasma
mem-
brane
(4).
Additionally,
studies
with
'5N
show
that
net
uptake
of
NO3-
is
largely
regulated
by
influx
rather
than
efflux
(20),
with
a
two-
to
threefold
stimulation
of
influx
following
24
h
of
NO3-
deprivation
of
previously
induced
roots.
However,
there
is
little
information
for
maize
on
changes
in
short-term
influx
kinetics
in
the
low
concentration
range
of
mechanism
1
(7)
for
plants
of
differing
N
status.
Use
of
'3N
as
a
tracer
for
such
studies
is
essential.
This
is
because,
firstly,
short
periods
of
labelling
are
required:
tracer
NO3-
can
equilibrate
rapidly
with
endogenous
pools
of
NO3-,
so
that
errors
in
estimating
influx
arise
if
there
is
appreciable
efflux
of
previously
absorbed
tracer
(17,
18).
Secondly,
influx
can
be
measured
at
low
external
concentrations
of
NO3-
(1
AiM
or
less)
using
cyclotron-
generated
'3N
of
high
specific
activity,
giving
a
precision
not
possible
with
'sN
enrichment.
The
purpose
of
the
present
study
with
'3N
was
to
charac-
terize
more
fully
the
NO3-
transport
system
in
maize
roots.
Specifically
we
measured
(a)
short-term
influx
of
NO3-
in
starved
and
unstarved
roots
as
a
function
of
the
external
concentration,
(b)
induction
of
NO3-
influx,
and
(c)
the
relation
between
total
N
concentration
in
roots
and
NO3-
influx.
MATERIALS
AND
METHODS
Growth
of
Plants
and
Experimental
Treatments
Hybrid
maize
(Zea
mays
cv
Pioneer
3906)
was
germinated
on
moist
paper
in
the
dark
in
an
incubator
at
25°C.
After
3
d,
seedlings
were
transferred
in
sets
of
4,
each
to
an
expanded
polystyrene
float
on
a
1
L
volume
of
a
dilute,
nutrient
solution
containing
(mM):
KH2PO4,
0.5;
MgSO4,
0.5;
Ca(NO3)2,
0.5;
Fe
EDTA,
50
,uM,
and
micronutrients.
The
temperature
was
24
to
26°C,
the
PPFD
was
300
,Amol
m-2s-'
with
a
15
h
daylength;
and
nutrient
solutions
were
continuously
bubbled
with
air.
After
8
d,
solutions
were
replenished,
either
with
the
same
solution,
or
with
one
lacking
N,
in
which
Ca(NO3)2
was
642

NITRATE
INFLUX
INTO
MAIZE
ROOTS
replaced
by
0.5
mm
CaSO4.
Kernels
were
removed
at
this
time
from
all
seedlings,
so
that
plants
became
dependent
on
the
nutrient
solution
for
supplies
of
mineral
nutrients.
After
60
h
of
N
deprivation,
fresh
nutrient
solution
containing
0.05
mM
Ca(NO3)2
was
supplied
to
both
the
N-starved
and
control
(unstarved)
plants.
Influx
was
measured
usually
6
h
after
the
resupply
of
NO3-.
However,
some
experiments
examined
the
effect
of
concentration
of
NO3-,
and
duration
of
exposure,
on
induction
of
NO3-
transport
(see
"Results").
For
studies
involving
inhibitors,
the
following
were
used
at
the
indicated
concentrations:
6-methylpurine
(0.5
mM),
phen-
ylglyoxal
(0.5
mM),
FITC2
(0.5
mM),
cyclohexmiade
(2
mg
L-')
and
chloramphenicol
(50
mg
L-').
Plants
were
exposed
to
inhibitors,
dissolved
in
the
complete
nutrient
solution,
for
60
min
(phenylglyoxal,
FITC)
or
6
h
(methylpurine,
cyclo-
heximide,
chloramphenicol)
before
measurement
of
NO3-
influx.
Preparation
and
Uptake
of
13N03-
The
radioisotope
'3N
was
generated
by
bombardment
of
pure water
with
a
proton
beam
(17.6
MeV
and
10-15
,uA
current)
in
a
recirculating
target
at
the
cyclotron
in
the
Posi-
tron
Diagnostic
and
Research
Center,
University
of
Texas
Health
Science
Center
at
Houston.
Nitrate
labeled
with
13N
was
purified
from
other
'3N
products
according
to
modified
procedures
of
Lee
and
Clarkson
(13),
but
with
inclusion
of
an
alumina
cartridge
(2
cm
x
3
mm
i.d.)
to
remove
18F
simultaneously
produced
from
180
(natural
abundance
0.2%).
Verification
of
purity
was
accomplished
by
HPLC,
which
gave
a
single
peak
for
'3NO3-,
and
by
the
half-life
of
radioac-
tive
decay.
The
'3N
radioisotope
was
added
to
the
uptake
solutions
containing
a
pH
6.0
'buffer'
(CaSO4
and
KH2PO4,
both
at
0.5
mM)
in
which
the
concentration
of
NO3-
was
from
1
to
250
,gM.
A
5
mL
sample
of
each
solution
was
counted
before
uptake
to
determine
specific
activity,
which
ranged
from
200
to
14000
Bq
nmol-'
NO3-.
Roots
of
seedlings,
supported
by
their
polystyrene
floats,
were
transferred
to
the
pH
6.0
buffer
for
two
periods
of
exactly
1
min
each
to
remove
any
free
space
NO3-.
The
floats
were
then
placed
in
the
uptake
solution
for
exactly
5
min
so
that
roots,
but
not
coleoptile
or
leaves,
were
in
contact
with
labeled
ions.
A
group
of
starved
and
un-
starved
seedlings
were
placed
in
each
of
the
uptake
solutions
at
the
same
time.
After
the
uptake
period,
the
roots
of
intact
seedlings
were
rinsed
again
in
fresh
pH
6.0
buffer
for
two
1
min
periods
and
placed
in
polyethylene
containers
to
be
counted.
Times
were
accurate
to
within
+
5
s,
and
all
solutions
were
vigorously
stirred
and
aerated
by
bubbling with
air.
Typically,
five
batches
of
'3N
were
prepared
each
experimental
day.
Measurements
of
influx
began
2
h
after
the
start
of
the
photoperiod.
Each
of
the
five
separate
experiments
contained
the
full
range
of
treatments,
so
that
any
effect
of
photoperiod
would
not
bias
treatment
effects.
2
Abbreviations:
FITC,
fluorescein
isothiocyanate;
Imax,
the
maxi-
mum
influx
at
saturating
concentrations
of
NO3-
in
the
low
concen-
tration
range
of
mechanism
I
(7).
Radioassay
of
13N
The
amount
of
"3N
in
the
plant
and
solution
samples
was
determined
by
use
of
a
NaI
crystal
scintillator
(100
mm
height
x
100
mm
diameter,
with
a
50
x
50
mm
center
well)
mounted
on
a
photomultiplier
tube
which
was
biased
with
1000
V
from
a
high
voltage
power
supply.
The
signal
was
processed
by
a
preamplifier
and
amplifier
connected
to
a
multi-channel
analyzer
(The
Nucleus
Inc.,
Model
PCA
4000)
controlled
by
a
micro-computer.
The
energy
levels
of
the
y-rays
resulting
from
annihilation
of
positrons
emmited
by
'3N
were
cali-
brated
using
standard
22Na,
57Co,
and
6Co
sources.
Samples
were
counted
for
30
s
with
dead
time
correction,
and
integra-
tion
of
peak
area
with
subtraction
of
background
was
per-
formed
by
the
multi-channel
analyzer
software.
All
counts
were
corrected
for
half-life
to
an
arbitrary
starting
point.
In
addition,
purity
of
'3N
in
solutions
and
plants
was
verified
by
monitoring
half-life
through
recounting,
and
by
y
energy.
In
all
experiments,
our
observed
half-life
and
y
energy
were
in
agreement
with
published
values
(9.97
min
and
0.511
MeV)
(10).
Nitrogen
Determination
Fresh
weights
and
dry
weights
of
roots
and
shoots
of
the
seedlings
were
recorded
and
samples
were
ground
for
total
N
analysis.
Total
N
concentration
was
determined
using
a
Per-
kin-Elmer
model
2400
elemental
analyzer.
RESULTS
AND
DISCUSSION
Uptake
with
Time
The
uptake
of
nitrate
from
a
100
,M
NO3-
solution
by
seedlings
was
examined
over
a
30
min
period.
The
labeled
nitrate
accumulating
in
both
roots
and
shoots
can
be
described
by
a
curvilinear
relationship
(Fig.
1).
There
was
a
slight
decrease
in
the
observed
rate
of
uptake
by
the
roots
beginning
2500
y4.O054+90.7(x)0.49(x2)
2000
Root
la
1500
.
1000
.
o
500
-
yu0074+719(x)+0.25(x
Shoot
0
0
7
14
21
28
35
Uptake
time
(min)
Figure
1.
Uptake
of
13N-labeled
NO3-
by
intact
maize
seedlings
with
time.
Plants
were
grown
with
nutrient
solution
containing
1
mm
nitrate
and
were
N-starved
for
3
d
followed
by
6
h
resupply
of
100
lM
NO3-
directly
before
measurement
of
uptake.
Each
value
is
mean
±
SE
for
four
determinations.
Equations
given
have
r2
=
0.97
(root)
and
r2
=
0.93
(shoot).
643

Plant
Physiol.
Vol.
93,
1990
around 20
min.
Efflux
of
nitrate
was
probably
responsible
for
this
apparent
decrease
in
influx,
as
appreciable
efflux
of
tracer
by
30
min
has
been
reported
for
barley
(13)
and
maize
(17).
At
the
same
time,
transport
of
NO3-
from
the
roots
to
the
shoots
increased
slightly.
The
slow
initial
transport
to
the
shoot
may
have
been
due
to
filling
of
cytoplasmic
and
vacu-
olar
pools
in
the
previously
N-starved
root
cells.
As
the
pools
were
filled,
more
nitrate
was
available
for
transport
to
the
shoot.
The
duration
of
'3N
uptake
for
all
other
experiments
was
5
min,
which
falls
well
within
the
linear
range
where
effects
of
efflux
on
estimates
of
influx
are
minimized.
Induction
Times
and
Concentrations
The
induction
of
increased
uptake
capacity
has
been
shown
to
be both
time
and
concentration
dependent
in
barley
(14,
15,
19).
We
investigated
the
time
required
for
complete
induction
in
starved
roots
of
maize.
Influx
was
measured
subsequently
at
50,
100,
and
250
jM
NO3-
after
0,
3,
6,
12,
and
24
h
of
incubation
with
100
,M
NO3-.
Influx
of
'3N03-
in
control
(unstarved)
seedlings
was
also
measured.
The
constitutive
influx
for
roots
of
seedlings
that
had
not
been
exposed
to
NO3-
for
at
least
3
d
was
generally
lower
than
in
seedlings
that
have
been
grown
continuously
in
a
solution
containing
NO3-
(Fig.
2).
Six
hours
of
contact
with
NO3-
(100
jAM)
were
required
to
maximize
influx
from
all
three
NO3-
concentrations
following
a
period
of
starvation.
A
leveling
off
or
decrease
in
influx
was
observed
after
12
h
of
induction.
This
may
have
been
due
to
a
sufficient
increase
in
the
N
concentration
within
the
plant
to
elicit
a
negative
regulation
of
the
transport
system.
Whether
this
occurs
through
accumulation
of
intracellular
NO3-,
or
a
product
of
its
metabolism,
is
debatable
(15,
19).
Nitrogen
concentrations
in
seedlings
used
in
this
study
rose
considerably
over
a
23
h
induction
period
(by
46%
for
roots
and
87%
for
shoots)
and
E
1
145
E
110
5
75
0
z
404
+N
0
5
10
15
20
25
Duration
of
inductioni
(h)
Figure
2.
Influx
of
13N-labeled
NO3-
by
intact
maize
seedlings
with
different
times
of
induction.
Seedlings
were
N-starved
(open
markers)
for
3 d
and
resupplied
with
100
ltM
NO3-
for
various
times
prior
to
measuring
influx.
Solid
markers
are
unstarved
controls.
Concentration
of
N03-
in
the
uptake
solution
were
at
the
levels
stated.
(A,
A;
250
AtM;
0,
*;
100
uM:
O,
*;
50
jAM)
Each
value
is
the
mean
±
SE
for
12
determinations
from
three
separate
experiments.
Table
I.
Concentration
of
N
in
Roots
and
Shoots
of
N-Starved
Seedlings
during
Exposure
to
100
tM
NO3
Duration
of
Root
Shoot
Induction
h
mmol
g-1
dry
wt
0
1.78
±
0.08a
1.82
±
0.14
3
1.75±0.01
2.63±0.10
6
2.01
±
0.04
2.52
±
0.06
12
2.36±0.16
2.88±0.10
23
2.61
±
0.04
3.11
±
0.01
+N
Control
2.46
±
0.33
3.40
±
0.05
a
Values
show
mean
±
SE
(n
=
4).
root
N
concentrations
actually
reached
those
of
unstarved
seedlings
(Table
I).
Different
concentrations
of
NO3-
(0,
5,
25,
50,
100
,uM
NO3-)
were
used
for
a
6
h
induction
period
of
N-starved
seedlings,
followed
by
measurement
of
influx
at
one
concen-
tration
(100
lM)
(Table
II).
Influx
was
correlated
(r2
=
0.97)
with
the
concentration
of
NO3-
used
for
induction.
Siddiqi
et
al.
(19)
found
in
barley
that,
generally,
lower
concentrations
of
NO3
used
for
induction
require
longer
periods
of
contact
to
achieve
maximum
influx
of
'3NO3-.
Our
data
parallel
closely
those
of
MacKown
and
McClure
(16)
who
found
induction
of
net
NO3-
uptake
was
maximal
at
6
to
8
h
after
continuous
or
discontuous
contact
with
NO3-.
Regulation
of
influx
in
maize
thus
is
similar
to
that
in
barley
although
at
least
12
to
24
h
of
induction
was
necessary
for
maximal
induction
in
barley
(19)
in
contrast
to
the
shorter
period
found
optimal
in
maize.
Kinetics
of
Uptake
The
influx
of
NO3-
by
unstarved
and
starved
seedlings
after
a
6
h
induction
period,
as
a
function
of
the
external
concen-
tration,
can
be
described
by
Michaelis-Menten
kinetics
(Fig.
3).
Estimates
of
Imax
for
fully
induced,
starved
seedlings
differ
by
a
factor
of
1.7
(159:94)
from
un-starved
ones.
This
corre-
sponds
closely
to
the
difference
found
by
Lee
and
Drew
(14)
in
barley
seedlings.
As
noted
here,
however,
the
initial
uptake
in
uninduced
starved
seedlings
was
lower
than
in
N-sufficient
plants,
and
the
Imax
difference
was
observed
only
after
a
6
h
induction
period.
By
contrast,
in
barley
(14),
differences
in
influx
between
plants
starved
for
3
d
and
unstarved
roots
were
apparent
immediately
upon
contact
with
'3N03-,
before
fur-
ther
induction.
Induction
for
5.3
h
greatly
accentuated
this
difference.
Similarly,
in
maize,
net
uptake
and
15NO3-
influx
(over
a
30
min
period)
were
enhanced
two-
to
threefold
by
temporary
deprivation
of
NO3-,
with
24
h
of
deprivation
inducing
a
maximum
stimulation,
at
25°C.
At
longer
times
of
NO3-
deprivation,
net
uptake
rate
and
influx
declined
to
about
the
initial,
unstarved
values
(20).
Influx
of
NO3
in
maize
roots
is
most
likely
dependent
on
the
activity
of
a
transport
protein,
but
at
present
it
can
only
be
assayed
in
vivo
and
not
in
purified
form,
so
that
interpre-
tation
of
Michaelis-Menten
kinetics
is
speculative.
Increase
in
Imax
may
be
brought
about
by
a
greater
turnover
number
of
HOLE
ET
AL.
644

NITRATE
INFLUX
INTO
MAIZE
ROOTS
Table
II.
Effect
of
Induction
with
Different
Concentrations
of
NO3
on
Influx
of
13NO3
Inductiona
conducnrtiona
Nitrate
lnflUXb
Concentration
Mm
NO3
nmol
g-'
fresh
wt
min'
0
55.8
±
6.2
5
57.1
±4.3
25
79.9
±
1.5
50
84.8
±
6.6
100
120.7
±
5.7
a
Induction
period
was
6
h.
b
Uptake
concentration
was
100
,gM
N03.
Values
show
mean
±
SE
(n
=
4).
the
existing
transport
proteins,
or
due
to
greater
synthesis
of
the
transport
protein
per
unit
weight
of
root.
The
observed
increase
in
Imax
following
starvation
may
then
be
explained
as
simply
a
production
of
more
of
the
same
transport
protein.
However,
this
is
incompatible
with
the
consistent
and
signif-
icant
differences
observed
in
Km
between
starved
and
unstar-
vedroots
(18.6
and
24.0
,M,
respectively).
These
Km
values
are
somewhat
smaller
than
those
observed
for
net
uptake
in
maize
(21)
but
as
has
been
noted
earlier
(14),
estimates
of
Km
for
net
uptake
measured
over
long
times
would
be
expected
to
be
larger
because
the
half-maximum
rate
would
have
to
include
efflux
as
well
as
influx.
The
implication
of
real
differ-
ences
in
Km
are
that
the
transport
proteins
differ
in
some
structural
(and
functional)
way
from
each
other
or
are
alloste-
rically
affected.
Allosteric
regulation
resulting
in
the
initial
decrease
in
Km
would
not
require
de
novo
protein
synthesis
as
observed
in
this
study,
though
it
may
play
a
role
during
the
phase
when
different
estimates
of
Km
are
observed
during
rising
root
[NO3-]
in
barley
(19).
By
contrast,
during
the
initial
increases
of
NO3-
influx
in
barley,
Km
values
estimated
under
comparable
conditions
of
solution
stirring
were
ap-
proximately
half
those
for
maize
and
unaffected
by
N
star-
vation
(14).
Influx
in
uninduced
roots
measures
only
the
constitutive
transport
system
(Fig.
2;
Table
II),
while
influx
in
induced
tissue
may
summate
the
constitutive
and
inducible
systems.
If
this
interpretation
is
correct,
Imax
in
induced
roots
is
the
addition
of
two
Imax
values,
while
the
estimate
of
Km
will
lie
between
the
two
separate
values.
Low
concentrations
of
exogenous
NO3-
induce
a
greater
increase
in
'3NO3-
influx
in
barley
(19)
than
in
maize
(Table
II),
apparently
without
any
threshold
level.
Enhanced
net
uptake
of
NO3-
(16)
was
likewise
induced
by
as
little
as
10
,uM
NO3-,
with
a
detectable
increase
at
2
h
although
maximal
induction
was
not
complete
at
12
h.
It
would
be
interesting
to
know
whether
this
behavior
reflects
a
concentration-de-
pendent
regulation
of
the
syntehsis
of
the
inducible
transport
system
at
the
transcription/translation
level
(23).
Alterna-
tively,
not
all
the
cortical
cells
in
the
root
will
be
exposed
uniformly
to
the
outer
solution.
At
higher
external
concentra-
tions,
NO3-
may
diffuse
further
through
the
root
apoplast,
exposing
a
greater
number
of
cortical
cells
to
NO3-,
raising
their
transport
capability.
Experiments
on
'3NO3-
influx
with
root
protoplasts
may
help
distinguish
between
these
possibil-
ities,
since
all
cells
would
be
uniformly
exposed
to
the
medium.
Characteristics
of
the
Inducible
Transport
System
Various
potential
inducers
(including
nitrate)
and
inhibitors
were
used
during
the
induction
and
influx
periods
to
isolate
events
that
are
necessary
for
full
induction
of
the
transport
mechanism.
Potential
inducers
were
used
for
6
h
in
place
of
the
normal
inducer
(NO31
in
starved
plants
and
were
added
to
the
nitrate
containing
solution
of
un-starved
plants
for
6
h
prior
to
influx
measurement
to
detect
possible
inhibitory
effects.
None
of
the
potential
inducers
other
than
nitrate,
were
effective
in
promoting
an
increase
in
influx
over
that
in
uninduced
N-starved
seedlings
(Table
IIIA).
Chloride,
am-
monium,
and
sulfate
had
no
significant
effect
on
influx
while
chlorate
and
nitrite
depressed
the
uptake
rate
compared
to
uninduced
roots.
In
roots
that
had
already
been
induced
(Table
IIIA,
left
column),
there
was
no
significant
inhibitor
effect
of
previous
exposure
to
potential
inducers,
so
that
the
capacity
to
transport
'3N03-
was
not
damaged
by
contact
with
any
of
the
salts.
A
range
of
inhibitors
was
used
to
examine
their
effect
on
induction
of
enhanced
'3NO3-
influx.
All
inhibitors
were
used
in
conjunction
with
100
gM
NO3-
as
an
inducer
in
starved
roots.
FITC
and
phenylglyoxal
bind,
respectively,
to
exposed
lysine
and
arginine
residues
in
plasma
membrane
proteins,
and
have
been
used
to
inhibit
specifically
the
transport
of
anions
(Cl-
and
NO3-)
in
erythrocytes
(1)
and
in
maize
roots
(5).
Influx
of
'3NO3-
was
strongly
inhibited
by
0.5
mm
FITC
and
by
0.5
mM
phenylglyoxal
(Table
IIIB),
becoming
equiv-
alent
to
the
binding
of
'3NO3-
by
dead
roots.
A
similar
inhibition
by
phenylglyoxal
of
net
uptake
of
NO3-
in
maize,
200
Oa
_-.
1
100
0
0
100
300
NO:3
concentration
in
solution
(uM)
Figure
3. Influx
of
13N-labeled
N03-
by
intact
maize
seedlings
from
uptake
solutions
of
differing
N03-
concentration.
Plants
were
grown
with
nutrient
solution
containing
1
mm
nitrate
and
were
N-starved
(0)
for
3
d
followed
by
resupply
for
6
h
with
100
gM
N03-,
or
were
unstarved
(0)
prior
to
uptake
measurement.
Each
value
is
mean
+
SE
for
11
determinations
from
four
separate
experiments.
Michaelis-
Menten
equations
(fitted
lines)
have
r2
=
0.81
(N-starved)
and
r2
=
0.79
(unstarved).
645

Plant
Physiol.
Vol.
93,1990
Table
Ill.
Induction
of
13NO-
Influx
in
Presence
of
Various
Potential
Inducers
and
Inhibitors
Concentration
of
13NO3
was
100
,uM,
for
5
min
influx.
Unstarved
(induced)
plants
had
been
maintained
in
solution
with
1.0
mM
NO-.
NO-
starved
(uninduced)
plants
were
starved
of
NO-
for
3
d.
Unstarved
N03-Starved
A.
Inducersa
(induced)b
(uninduced)c
nmol
g-l
fresh
wt
min-'
KCI03
70.0±
14.2(n=4)
33.0±4.4(n=8)
KCI
76.0
±
16.1
80.0
±
3.7
(NH4)2SO4
64.0
±
6.9
74.0
±
4.4
KNO2
69.0
±
9.1
30.0
±
7.7
KNO3
79.0
±
9.0
110.0
±
9.4
None
58.0
±
3.4
Sd
Unstarved
N03-Starved
B.
Inhibitorsd
(induced)b
(induced)b
nmol
g'
fresh
wt
min-'
6-Methyl
purine
ND
(n
=
4)
47.0
±
4.3
(n
=
4)
Cycloheximide
40.0
±
0.1
33.0
±
3.5
Chloramphenicol
80.0
±
2.3
120.0
±
11.5
Phenylglyoxal
ND
9.6
±
1.3
FITC
ND
6.9
±
0.3
None
99.0
±
5.7
101.0
±
9.4
Killed
rootse
ND
4.1
±
0.5
a
Inducers
were
all
at
100
MAM,
supplied
for
6
h
before
measurement
of
13NO3
influx,
but
not
present
during
influx.
b
During
6
h
period,
the
basal
solution
contained
100
AM
NO3.
c
During
6
h
induction
period,
and
for
3
d
prior,
basal
solutions
were
NO--free.
d
Inhibitors
were
supplied
for
1
to
6
h
before
measuring
influx,
and
included
in
the
uptake
medium
(see
"Materials
and
Meth-
ods").
e
Killed
by
1
min
microwave
exposure.
measured
by
depletion
of
nitrate
from
the
uptake
solution
at
hourly
intervals,
was
reported
(5),
but
a
lack
of
inhibition
by
FITC
reported
by
the
same
authors
contrasts
with
our
results.
Our
findings
raise
doubts
about
the
ability
of
these
two
inhibitors
to
distinguish
between
plasma
membrane
anion
transporters
of
different
specificities
in
maize:
it
seems
prob-
able
that
both
anion
transporters
are
sensitive
to
FITC,
as
occurs
in
red
blood
cells
(1).
Roots
of
maize
used
in
the
study
by
Dhugga
et
al.
(5)
were
cold-shocked,
before
measuring
net
uptake,
and
differences
between
our
results
may
be
related
to
that
pretreatment.
Chloramphenicol
inhibits
translation
in
ribosomes
of
mi-
tochondria
and
plastids,
but
does
not
affect
that
in
cytosolic
80S
ribosomes.
Roots
treated
with
chloramphenicol
for
the
length
of
the
induction
period
were
as
capable
of
uptake
as
fully
induced
control
roots
(120
and
101
nmol
NO3-
g-'
fresh
weight
min-'
respectively,
Table
IIIB).
By
contrast,
cyclohex-
imide
inhibits
translation
in
cytosolic
ribosomes,
and
influx
in
cyclohexamide
treated
roots
remained
identical
to
the
uninduced
level,
despite
exposure
to
NO3-
(33
and
58
nmol
NO3-
g-'
fresh
weight
min-'
respectively).
This
result
is
comparable
to
that
found
with
long-term
net
uptake
(11)
and
provides
some
evidence
that
there
is
a
translational
event
involving
nuclear
coded
mRNA
for
the
complete
induction
of
the
transport
protein.
When
transcription
was
interrupted
with
6-methylpurine,
influx
was
again
only
as
high
as
in
the
uninduced
controls
(47
and
58
nmol
NO3-
g-'
fresh
weight
min-'
respectively,
Table
III),
agreeing
with
earlier
results
on
net
uptake
(11).
It
should
be
noted
that
rates
of
influx
in
the
6-methylpurine
and
cyclohexamide
treatments
did
not
de-
cline
compared
to
uninduced
(constitutive)
controls
providing
some
indication
of
membrane
integrity
and
normal
operation
of
the
constitutive
transport
system
(Table
III).
Clearly,
reg-
ulation
of
induction
of
the
transport
system
must
be
at
the
DNA
level.
Together,
these
data
are
consistent
with
a
transport
sys-
tem(s)
that
is
regulated
at
the
transcriptional
level
by
the
concentration
of
nitrogen
in
the
plant
and
its
immediate
environment.
GENERAL
DISCUSSION
The
present
results
show
that
in
maize,
as
in
barley
(14,
19)
the
constitutive
influx
of
NO3-
is
low
in
roots
not
recently
exposed
to
nitrate.
We
propose
that
this
uninduced
condition
be
designated
state
1
for
NO3-
transport.
Influx
is
greatly
increased
in
NO3--induced
roots
(state
2),
a
response
specific
to
NO3-
exposure,
reaching
a
maximum
some
6
h
after
initial
contact
with
NO3-.
In
terms
of
Michaelis-Menten
kinetics,
the
Imax
and
possibly
the
Km
are
regulated
by
the
N
status
of
the
roots,
such
that
in
N-starved
roots
the
inducible
compo-
nent
is
further
enhanced
(state
2E).
State
2E
appears
to
be
transient
(19,
20),
its
expression
declining
as
the
N
status
of
root
and
shoot
increase
following
NO3-
feeding,
to
the
state
2
level
found
in
plants
continuously
maintained
on
NO3-.
The
transition
from
state
1
to
state
2
is
apparently
dependent
on
nuclear
DNA
coded
RNA
synthesis,
and
on
cytoplasmic
protein
synthesis
(Table
III;
see
also
ref.
11).
The
inducible
transport
system
for
NO3-
thus
may
be
controlled
by
repres-
sion/derepression
at
the
DNA
level.
The
extent
to
which
state
2E
exceeds
state
2
appears
to
be
under
feed-back
regulation
by
the
concentration
of
N
within
the
root.
Whether
this
regulation
is
by
cytoplasmic
NO3-
concentration,
or
by
a
reduced
product
of
NO3--metabolism
has
been
discussed
by
Lee
and
Rudge
(15)
and
by
Siddiqi
et
al.
(19).
During
tem-
porary
deprivation
of
NO3-,
the
induced
state
2
is
lost
(de-
induction)
(19)
at
a
rate
that
is
highly
variable
for
different
species
and
conditions,
reflecting
perhaps
the
depletion
of
cytoplasmic
and
vacuolar
NO3-
in
roots
through
transport
to
growing
tissues
of
the
plant.
Although
mutant
anlaysis
in
barley
has
shown
that
N03-
transport
is
mediated
by
genetic
loci
that
are
distinct
from
those
for
nitrate
reductase
and
nitrite
reductase
(22),
the
simultaneous
induction
of
state
2
and
these
enzymes
suggests
operation
of
a
regulatory
network
controlling
N
acquisition
and
metabolism,
analogous
to
that
in
microbial
systems
(23).
The
similarity
between
changes
in
influx
of
3N03
in
recent
reports
(14,
19)
and
longer
term
measurements
of
net
uptake
(11,
15,
20)
suggests
that
regulation
of
NO3-
movement
into
roots
is
principally
by
influx,
and
not
by
efflux.
Similar
conclusions
were
reached
by
Teyker
et
al.
(20)
from
study
of
15N03-
uptake
and
'4NO3-
leakage,
and
contrasts
to
an
earlier
report
(9)
that
efflux
regulated
NO3-
net
uptake.
However,
there
was
a
detectable
efflux
of
'4NO3-
(into
'5N03-
solutions)
from
maize
roots
(20),
its
extent
being
proportional
to
root
646
HOLE
ET
AL.

NITRATE
INFLUX
INTO
MAIZE
ROOTS
tissue
NO3-
concentration
(mainly
vacuolar),
an
efflux
that
declined
with
increasing
exposure
to
N-free
solutions.
ACKNOWLEDGMENTS
The
technical
assistant
of
P.
Bethke,
P.
Hole,
B.
Jez,
J.
Johnson,
and
S.
Pezeshgi
is
greatly
appreciated.
Our
thanks
also
to
Mr.
and
Mrs.
W.
G.
Hole
of
Houston
for
accommodation.
We
also
thank
Pioneer
Seed
Co.
for
supplies
of
cv
3906.
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