Metabolism of nitrate- and ammonium-nitrogen absorbed from ammonium nitrate and ammonium sulfate in the tobacco plant


Yoshida, D

Soil Science and Plant Nutrition 12(3): 1-5

1966


SOIL
SCIENCE
AND
PLANT
NUTRITION
VOLUME
12
May,
1966
NUMBER
3
METABOLISM
OF
NITRATE-
AND
AMMONIUM-NITROGEN
ABSORBED
FROM
AMMONIUM
NITRATE
AND
AMMONIUM
SULFATE
IN
TIIE
TOBACCO
PLANT
Daisuke
YosiDDA
llatano
Tobacco
Experiment
Station,
Japan
Monopoly
Corporation
RECEIVED
JANUARY
12,
1965
In
water
culture
experiments
with
tobacco
plants,
ammonium
nitrate
has
been
generally
used
as
a
nitrogen
source.
In
a
comparison
among
various
nitrogen
sources
for
water
cultured
tobacco,
better
growth
has
been
obtained
with
ammonium
nitrate
than
with
sodium
nitrate
and
ammonium
sulfate
(1).
It
has
been
shown
that
both
ammonium-
and
nitrate-nitrogen
are
simultaneously
absorbed
by
the
tobacco
plant
from
ammonium
nitrate
(2).
It
has
been
also
apparent
that
physio-
logical
toxicity
developed
when
ammonium-
nitrogen
was
absorbed
from
ammonium
sulfate.
It
has
been
considered
that
this
toxicity
developed
by
the
abnormal
metabo-
lism
of
ammonium-nitrogen
in
the
plant.
No
toxicity
appears
to
develop
when
ammonium-
nitrogen
is
absorbed
from
ammonium
nitrate.
In
the
present
paper,
the
metabolism
of
ammonium
-
and
nitrate-nitrogen
absorbed
from
ammonium
nitrate
and
ammonium
sulfate
in
the
tobacco
plant
was
investigated.
Materials
and
Methods
Culture
of
plants
.
Tobacco
plants
(Nicotiana
tabacum
var.
Bright
Yellow)
were
grown
in
a
culture
solution
containing
40
ppm
of
nitrogen,
20
ppm
phosphate,
80
ppm
each
of
potassium
and
calcium,
10
ppm
magnesium,
1
ppm
iron,
0.1
ppm
each
of
manganese
and
zinc,
0.05
ppm
boron,
0.01
ppm
each
of
cupper
and
molybdenum.
Culture
was
carried
out
in
the
green
house.
Treatments
by
"N-labeled
culture
solution:
Culture
solutions
containing
40
ppm
of
nitrogen
as
ammonium
nitrate
consisted
of
either
"N-labeled
a
mmonium-natural
nitrate
or
natural
ammonium-
1
"N-labeled
nitrate
(in
the
later
part
of
this
paper,
these
labelings
will
be
mentioned
as
reciprocally
labeled
ammonium
nitrate)
and
"N-
labeled
ammonium
sulfate
were
supplied
after
the
plants
had
been
grown
for
14
days
(12
leaf
stage).
Culture
solutions
containing
the
labeled
nitrogen
were
supplied
from
9
A.M.
to
2
P.M.
on
12th.
November,
1963.
The
day
was
fine
and
the
temper-
ature
in
the
green
house
was
about
25°C.
Ammonium
nitrate
containing
"N-labeled
ammonium-nitrogen
was
prepared
by
the
distillation
of
ammonium
sulfate
containing
"N
as
39.6
atom
excess
per
cent
with
alkali
and
collecting
in
nitric
acid.
Ammonium
nitrate
containing
"N-labeled
nitrate-nitrogen
was
prepared
by
neutralization
of
nitric
acid
containing
"N
as
39.6
atom
excess
per
cent
with
ammonium
hydroxide.
Concentration
of
"N
in
the
ammonium
sulfare
was
39.6
atom
exess
per
cent.
Eight
plants
were
employed
for
each
treatment.
Plants
were
harvested
after
5
hours
growth
in
the
culture
solution
containing
labeled
nitrogen,
divided
into
leaves
and
root
and
immediately
extracted
with
hot
0.5
per
cent
acetic
acid
solution.
Methods
of
analysis:
(1)
Protein-nitrogen;
Nitogen
in
the
residue
obtained
by
acetic
acid
extraction
was
determined
by
KJELDAHL's
method
and
ex-
pressed
as
protein-nitrogen.
(2)
Ammonium-nitrogen
:
Permutit
was
added
to
an
aliquot
of
the
extract
and
the
mixture
was
shaken
for
10
minutes
in
a
shaking
apparatus
for
the
adsorption
of
free
ammonium-nitrogen.
After
removal
of
the
liquid
phase,
the
ammonium-nitrogen
in
the
permutit
was
distilled
with
alkali.
(3)
Alkaloid-nitrogen
:
After
removal
of
ammonium-
nitrogen,
as
above,
alkaloid
was
precipitated
from
the
liquid
phase
by
silicotungustic
acid.
Alkaloid-
nitrogen
in
the
precipitate
was
determined
by
KJELDAHL'S
method.
(
85
)
D.
YOSHIDA
(4)
Amide-nitrogen:
The
filtate
from
the
alkaloid
precipitation
was
concentrated
to
a
small
volume
to
make
about
1
N
concentration
of
hydrochloric
acid
and
then
hydrolized
in
a
boiling
water
bath
for
2
hours.
Amide-nitrogen
was
determined
by
distillation
of
the
hydrolyzate
with
excess
alkali.
(5)
Nitrate-nitrogen:
Nitrate-nitrogen
was
deter-
mined
by
the
distillation
of
the
solution
remaining
from
the
amide-nitrogen
determination,
with
the
addition
of
DEvALDA's
alloy.
(6)
Residue-nitrogen
:
Nitrogen
in
the
solution
remaining
from
the
distillation
for
nitrate
determi-
nation
was
determined
by
KJELDAHL's
method.
(7)
"N-Abundance
in
the
nitrogen:
Using
the
am-
monium-nitrogen
in
the
distillate
made
by
the
de-
termination
of
each
nitrogen
fraction,
"N-abundance
was
measured
by
a
mass-spectometer
(HITACHI
RMI-2).
Results
and
Discussion
(1)
SW-Atom
excess
per
cent
in
the
nitrogen
i)
Root
As
shown
in
Table
1,
in
the
roots
of
plants
supplied
with
reciprocally
labeled
ammonium
nitrate,
15
N-atom
excess
per
cent
of
protein-,
ammonium-,
alkaloid
amide-
plus
residue-
nitrogen
were
appreciably
higher
when
the
ammonium-nitrogen
was
labeled
than
when
the
nitrate-nitrogen
was
labeled.
This
would
indicate
that
the
assimillation
of
nitrate-
nitrogen
was
slower
than
that
of
ammonium-
nitrogen
in
the
root.
The
15
N-atom
excess
per
cent
of
nitrate-nitrogen
in
the
plant
supplied
with
labeled
nitrate-nitrogen
and
of
ammonium-nitrogen
in
the
plant
supplied
with
labeled
ammonium-nitrogen
were
re-
spectively
highest
among
the
nitrogen
frac-
tions
of
the
root.
Since
the
content
of
nitrate-
nitrogen
was
higher
than
that
of
ammonium-
nitrogen
as
shown
in
Table
2,
labeled
nitrate-
nitrogen
was
diluted
in
greater
extent
with
the
non-labeled
nitrate-nitrogen
already
present
in
the
plant
at
the
beginning
of
the
treatment
than
was
labeled
ammonium-nitrogen.
There-
fore,
the
content
of
labeled
nitrate-nitrogen
was
not
lower
than
that
of
ammonium-nitrogen
in
the
root
as
Is
indicated
below.
Labeled
nitrogen
was
supplied
at
the
same
15
N-atom
excess
per
cent
in
each
treatment.
Since
equal
amounts
of
labeled
and
non-
labeled
nitrogen
were
contained
in
the
am-
monium
nitrate,
the
15
N-atom
excess
per
cent
In
the
total-nitrogen
of
ammonium
X
86)
Table
1.
"N-atom
excess
per
cent
in
each
nitrogen
fraction
in
tobacco
plants.
Nitrogen
fractions
Nitrogen
sources
NH
4
"NO.
"NILNO
g
(
15
NI
1
4) s04
Leaves
Protein-N.
0.21
0.13
0.08
Ammonium-N.
1.28
0.58
0.94
Nitrate-N.
1.00
0.03
0.02
Alkaloid-N.
0.07
0.04
0.08
Amide-N.
1.26
1.93
1.10
Residue-N.
1.46
0.85
1.16
Roots
Protein-N.
0.05
0.25
0.34
Ammonium-N.
3.4
18.6
21.0
Nitrate-N.
7.9
0.2
0.2
Alkaloid-N.
0.09
0.35
0.44
Amide-N.
Residue-N.
1
0.39
1.35
2.60
nitrate
was
half
compared
with
ammonium
sulfate.
It
is
therefore
probable
that
the
tsN.
atom
excess
per
cent
in
the
protein-,
am-
monium-
and
amide-
plus
residue-nitrogen
were
higher
in
the
root
of
the
plant
supplied
with
ammonium
sulfate
than
that
with
am-
monium
nitrate.
This
difference
in
the
protein-,
ammonium-
and
alkaloid-nitrogen
was
sm
a
ll
compared
with
amide-
plus
residue
-
nitrogen.
Great
differences
in
the
amide
plus
residue-
nitrogen
would
suggest
that
the
compon
ents
responsible
for
ammonium
toxicity
may
b
e
contained
in
this
fraction.
ii)
Leaves
There
were
great
differences
between
the
15
N-atom
excess
per
cent
in
the
nitrogen
fraction
of
the
leaves
and
that
of
the
root
s
.
As
shown
in
Table
1,
in
the
leaves
of
the
plants
supplied
with
recipracally
labeled
am-
monium
nitrate,
15
N-atom
excess
per
cent
of
protein-,
ammonium-,
nitrate-,
alkaloid-
and
residue-nitrogen
in
the
15
N-labeled
nitrat
e
..
treatment
was
higher
than
that
of
the
'5N.
labeled
ammonium-treatment.
With
the
e
x
.
ception
of
nitrate-nitrogen,
this
relation
was
the
opposite
to
the
site
of
the
root.
Thi
s
fact
would
indicate
that
the
amount
o
f
nitrate-nitrogen
absorbed
from
ammonium
nitrate
was
translocated
more
abundantly
to
the
leaves
from
the
root
and
metabolized
in
the
leaves
than
ammonium-nitrogen.
A
very
low
atom
excess
per
cent
of
15N
was
detected
in
the
nitrate-nitrogen
in
the
2
METABOLISM
OF
NITRATE-
AND
AMMNIUM-NITROGEN
IN
THE
TOBACCO
PLANT
Table
2.
Contents
of
total-
and
"N-labeled-nitrogen
in
tobacco
plants.
(per
cent,
dry
matter
basis)
Nitrogen
fraction
Nitrogen
sources
NH
4
16
N0,
'
5
N114N08
("
NH4)
2S
O
4
Total-N
Labeled-N
Total-N
Labeled-N
Total-N
Labeled-N
Leaves
Protein-N.
3.28
0.0174
3.36
0.0110
3.36
0.0068
Ammonium-N.
0.050
0.0016
0.043
0.0006
0.047
0.0011
Nitrate-N.
0.23
0.0058
0.24
0.0002
0.23
0.0001
Alkaloid-N.
0.140
0.0002
0.155
0.0001
0.120
0.0002
Amide-N.
0.082
0.0026
0.070
0.0034
0.078
0.0022
Residue-N.
0.52
0.020
0.60
0.013
0.41
0.012
Roots
Protein-N.
3.04
0.004
3.22
0.020
3.13
0.027
Ammonium-N.
0.18
0.015
0.16
0.075
0.16
0.085
Nitrate-N.
0.56
0.112
0.51
0.003 0.303
0.002
Alkaloid-N.
0.047
0.0001
0.051
0.0005
0.043
0.0005
Amide-N.
1
Residue-N.
0.91
0.009
0.96
0.033
1.06
0.070
root
and
leaves
of
the
plants
supplied
with
labeled
ammonium-nitrogen.
This
would
indicate
that
there
was
little
oxidation
of
ammonium-
nitrogen
to
nitrate-nitrogen
in
the
plant.
The
15
N-atom
excess
per
cent
of
the
protein-,
and
amide-nitrogen
were
higher
and
those
of
ammonium-,
alkaloid-
and
residue-nitrogen
were
lower
in
the
leaves
of
the
plant
supplied
with
ammonium
nitrate
than
that
with
am-
monium
sulfate.
Since
the
concentration
of
labeled
nitrogen
in
the
culture
solution
con-
taining
ammonium
sulfate
was
twice
that
of
ammonium
nitrate,
as
indicated
above,
15
N-atom
excess
percent
in
the
plant
can
be
expected
to
be
higher
than
that
of
ammonium
nitrate.
In
spite
of
this
relation,
decreases
in
15
N-atom
excess
per
cent
in
the
protein-
and
amide-
nitrogen
were
recorded
in
the
plant
supplied
with
ammonium
sulfate.
This
fact
may
due
to
the
decreased
rate
of
nitrogen
assimilation
with
the
supply
of
ammonium
sulfate.
(2)
Contents
of
total-
and
15
N-labeled-nitrogen
As
shown
in
Table
2,
there
was
little
difference
In
the
total-nitrogen
of
each
nitrogen
fraction
between
treatments
with
the
different
sources
of
labeled
nitrogen.
This
would
be
attributable
to
the
fact
that
the
different
sources
of
nitrogen
were
supplied
for
only
a
short
period
of
time.
There
were
great
differences
in
the
contents
of
15N-labeled
nitrogen
of
each
fraction
among
the
plants
supplied
with
the
different
sources
of
labeled
nitrogen.
To
discuss
the
results
more
easily
the
distribution
ratios
of
labeled
nitrogen
in
each
nitrogen
fraction
were
calculated
as
follows.
(3)
15
N-Distribution
among
the
various
nitrogen
fractions
i)
Root
In
the
roots,
as
shown
in
Table
3,
the
labeled
nitrogen
was
contained
at
the
highest
percentage
in
the
nitrate-nitrogen
when
NH
4
15
N0
8
was
supplied,
and
at
the
highest
percentage
in
the
ammonium-nitrogen
when
15
NH
4
N0
8
was
supplied.
This
would
be
attributable
to
the
fact
that
the
plants
were
cultured
with
labeled
nitrogen
for
a
short
period
of
time
and
were
harvested
when
the
labeled-nitrogen
was
being
supplied.
In
the
roots,
about
80
per
cent
of
labeled-nitrogen
was
contained
in
the
nitrate-
nitrogen
when
NH
4
15
N0
8
was
supplied,
and
57
per
cent
of
labeled-nitrogen
was
contained
in
the
ammonium-nitrogen
when
15
NH
4
N0
8
was
supplied.
That
is,
in
the
root,
nitrate-nitrogen
was
more
slowly
metabolized
than
ammonium-
nitrogen.
Although
there
was
little
difference
between
ammonium
nitrate
and
ammonium
sulfate
in
the
percentage
of
labeled
protein-
and
alkaloid-nitrogen
in
the
total-labeled-
nitrogen
of
the
root,
the
per
cent
of
amide
plus
residue-nitrogen
was
higher
in
the
latter.
This
would
suggest
also
the
presence
of
a
component
responsible
for
ammonium
toxicity
3
(
8Z
)
Nitrogen
fractions
Nitrogen
sources
NI-1
4
16
NO.
"Nli,NO.
("NH,
)
2
80
4
Leaves
Protein-N.
Ammonium-N.
Nitrate-N.
Alkaloid-N.
Amide-N.
Residue-N.
Total
Roots
Protein-N.
Ammonium-N.
Nitrate-N.
Alkaloid-N.
Amide-N.
1
Residue-N.
j
Total
36.6
38.8
30.2
3.4
2.1
4.9
12.2
0.7
0.5
0.4
0.4
0.9
5.4
12.0
9.9
42.0
46.0
53.6
100
100
100
2.9
15.2
14.6
10.7
57,0
46.1
79.8
2.2
1.0
0.2
0.4
0.3
6.4
25.2
38.0
100
100
100
D.
YOSHIDA
Table
3.
"N-distribution
among
nitrogen
fractions
in
tobacco
plants.
in
this
fraction.
ii)
Leaves
There
was
little
difference
in
the
per
cent
of
protein-,
ammonium-,
and
residue-nitrogen
in
the
total-labeled-nitrogen
of
the
leaves
between
the
plants
supplied
with
the
recipro-
cally
labeled
ammonium
nitrate.
That
is,
the
labeled
nitrogen
absorbed
from
both
the
supplies
of
reciprocally
labeled
ammonium
nitrate
was
incorporated
to
almost
the
same
extent
into
the
protein-
and
residue-nitrogen
which
occupied
about
80
per
cent
of
the
labeled-
nitrogen
in
the
leaves.
It
was
evident
that
the
nitrate-nitrogen
was
reduced
rapidly
and
as-
similated
almost
equally
in
the
leaves
with
the
ammonium-nitrogen.
In
the
leaves
of
the
plant
supplied
with
labeled
nitrate-nitrogen,
about
12
per
cent
of
the
labeled-nitrogen
was
contained
in
the
nitrate-nitrogen.
Whereas
in
the
leaves
of
the
plant
supplied
with
labeled
ammonium-nitrogen,
about
2
per
cent
of
the
labeled-nitrogen
was
contained
in
the
am-
monium-nitrogen.
This
would
be
attributable
to
the
fact
that
the
upward
movement
of
nitrate-nitrogen
was
rapid
compared
with
ammonium-nitrogen
as
described
below.
Labeled
ammonium-nitrogen
was
distributed
at
a
higher
percentage
in
the
ammonium-,
alkaloid-
and
residue-nitrogen
when
it
was
absorbed
from
ammonium
sulfate
than
from
ammonium
nitrate.
That
is,
it
was
clear
that
Table
4.
Distribution
of
"N-labeled
nitrogen
among
each
leaves
and
roots.
Plant
parts
Nitrogen
sources
NI
-
1
4
16
N°.
16
NI
-
1.N%
("NH4)
9
SO4
Nmg
Ratio
Nmg
Ratio
Nmg
Ratio
Leaves
Roots
Total
0.71
0.44
1.15
62
38
100
0.37
0.37
0.74
50
50
100
0.30
0.54
0.84
36
64
100
the
incorporation
of
ammonium-nitrogen
into
protein-nitrogen
decreased
and
that
into
free
ammonium-nitrogen
increased
in
the
leaves
when
ammonium-nitrogen
was
absorbed
from
ammonium
sulfate
compared
with
ammonium
nitrate.
This
phenomenon
may
be
related
to
the
ammonium
toxicity
which
developed
on
the
plants
supplied
with
ammonium
sulfate.
(4)
Distribution
of
labeled-nitrogen
among
th
e
leaves
and
roots
As
shown
in
Table
4.
1.15
mg
and
0.74
mg
of
labeled-nitrogen
were
absorbed
from
nitrate-
labeled
ammonium
nitrate
and
ammonium-
labeld
ammonium
nitrate
respectively.
Di
s
-
tribution
of
the
labeled
nitrogen
through
ou
t
the
plants
showed
a
rapid
translocation
o
f
nitrate-nitrogen
compared
with
ammonium-
nitrogen.
Although
1.89
mg
(1.15+0.74)
of
nitrogen
should
be
absorbed
from
ammonium
nitrate
when
both
ammonium-
and
nitrate-nitrogen
were
labeled
in
one
treatment,
only
0.84
rug
of
labeled
nitrogen
was
absorbed
from
a
m
-
monium
sulfate.
Translocation
of
labeled
nitrogen
to
the
leaves
from
the
root
was
less
when
ammonium-nitrogen
was
absorbed
from
ammonium
sulfate
than
from
ammonium
nitrate.
Decrease
in
the
upward
movement
of
ammonium-nitrogen
in
the
tobacco
plant
have
been
reported
previously
by
the
author(3).
Since
the
period
of
ammonium-nitrogen
supply
was
too
short
to
develop
ammonium
toxicity,
there
was
little
difference
between
the
incorporation
and
translocation
of
labeled
ammonium-nitrogen
absorbed
from
ammoniu
m
sulfate
and
from
ammonium
nitrate.
If
labeled
ammonium-nitrogen
were
supplied
to
a
plant
which
has
been
previously
cultured
with
am-
monium
sulfate,
the
difference
would
be
more
apparent.
(
88
)
4
EMTABOLISM
OF
NITRATE-
AND
AMMNIUM-NITROGEN
IN
THE
TOBACCO
PLANT
The
author
expresses
his
thanks
to
Dr.
E.
G.
BOLLARD,
Fruit
Reseach
Division,
Department
of
Scientific
and
Industrial
Research,
New
Zealand,
for
help
in
the
preparation
of
this
manuscript.
Summary
Incorporation
into
the
various
nitrogen
fractions
of
tobacco
plants
of
15
N-ammonium-
and
15
N-nitrate-nitrogen
absorbed
from
am-
monium
nitrate
labeled
reciprocally
with
15
N
and
ammonium
sulfate
labeled
with
15
N
was
studied.
1.
In
the
root,
the
15
N-atom
excess
per
cent
of
protein-,
ammonium-,
alkaloid-
and
amide
plus
residue-nitrogen
and
the
distribution
ratios
of
these
fractions
among
the
total-
labeled
nitrogen
were
appreciably
higher
in
the
plants
supplied
with
labeled
ammonium-
nitrogen
than
that
with
nitrate-nitrogen.
This
would
suggest
that
the
nitrate-nitrogen
in
the
tobacco
roots
was
more
slowly
assimilated
than
is
ammonium-nitrogen.
2.
Nitrate-nitrogen
absorbed
from
ammonium
nitrate
was
more
rapidly
translocated
to
the
leaves
than
ammonium
nitrogen,
and
as-
similated
almost
equaly
with
ammonium-
nitrogen
in
the
leaves.
3.
Incorporation
of
ammonium-nitrogen
into
protein
decreased
and
that
into
free
ammonium-
nitrogen
increased
in
the
leaves
when
it
was
absorbed
from
ammonium
sulfate
rather
than
ammonium
nitrate.
Literature
Cited
1)
TSUKADA,
H.
and
NISHIYAMA,
S.,
Sci.
Paper
Central
Res.
Inst.
Japan,
62,
1-38
(1936)
2)
TAKAHASHI,
T.,
Bull.
Ilatano
Tobacco
Expt.
Sta.,
50,
1-95
(1962)
3)
YOSHIDA,
D.
and
TAKAHASHI,
T.,
Soil
Sci.
Plant
Nutr.,
7,
157-164
(1961)
5
(
89
)