Proline
as
r I
E.A. Britikov
Botanisch
a source
of nitrogen
metabolism
in plant
J.
Schrauwen
and
H.F.
Linskens
Laboratorium, Universiteit, Nijmegen
SUMMARY
Branches
48
hrs
Chenopodium album. including
of
solution
15
of
of exposure
bound
in order
N-L-proline
amino
number
of
chlorophyll
and
amino-lipids
a
nitrogen
in
acids, especially
and
leaves
to absorb
the
fractions
the
is
Proline
15
N
and
considered
be
to
was
found
in free
well
as
important
an
a
After
stream.
acid
aspartic
put into
were
transpiration
separated.
were
arginine, alanine, glutamic
fraction.
inflorescences,
young
latter with
and
as
in
source
of
nitrogen.
1.
INTRODUCTION
A
large
to
their intensive
1959;
proline
growth,
as,
was
Schrauwen
2%
about
and
1966),
Britikov
1964a;
&
Proline
1969).
of
especially
Musatova
al.
et
and
local
some
in
growing
in
content
that
1966)
is
proline
a
and d.
active
as
but
proteins,
b.
potent
a.
as
a
source
proline
Perdrizet & Macquire
1962;
The
experiment presented
trogen. According
nously
2.
15
15
applied
MATERIAL
species
some
chlorophyll
1963;
1969).
It
was
assumed
incorporation
of energy,
c.
a
to
as
a
Microbiological
Visiting
of the
Acta
which
15
mg
were
N).
contamination
scientist from K.
USSR,
hypothesis
synthesis (Duranton
proline
as
a
possible
nitrogen
& Maille
1966).
source
atom
direct confirmation of this idea
album
Chenopodium
weight)
(5.73
of
source
of ni-
from exoge-
was
expected.
METHODS
branches of
N-L-proIine
be
into structural
mediator of metabolism of the
the distribution of the labelled
N-L-proline,
(Briti-
material which
may
Breyhan & Heilinger
here deals with
&
reaches
decreases after initiation
N-L-proline (Schwarz Bioresearch, Inc., 98.9% purity, 95%
applied
1
to
AND
20 g total fresh
15
utilized for
may be
al.
et
1966, Linskens
dormant and survival tissues. Some data obtained are in favour of the
that
al.
et
Shvedskaja &
1964; Britikov
of
reserve
for direct
prior
structures
(Breyhan
1963),
Tupÿ
sharply
possible physico-chemical
a
&
pollen grains
& Schrauwen
utilized in many ways, e.g.,
biologically
nitrogen,
Linskens
plant
points
pollen grains (Tupÿ
dry weight (Bathurst 1954),
1965,
completely
in
1964b;
pollen germination (Linskens
kov
found in
for instance,
Duranton & Maille 1962, Durzan & Steward
Kruzhilin
of
of free
amount
A.
was
Timiryazev
N
excess)
was
(young inflorescences with 3-4 leaves,
put into distilled
Two
15
parallel
water
containing
experiments
prevented
by
Institute of Plant
were
addition
of
50 mg
carried
penicillin
Physiology, Academy
of
out.
(20
of Sciences
Moscow.
Bot. Need.
19(4), August
1970
515
A.
E.
units/ml)
and
streptomycin
(natural day length)
the
total 48
hrs of the
were
carefully
washed
according
Scheme
to
Amino
the
liquid
nitrogen,
LINSKENS
of exposure
absorbed;
was
portions
during
of tap
the basal parts of the
of the
traces
hrs
H. F.
water
branches
proline solution, subsequently
and
homogenized
fractionated
scheme I.
acids
were
separated
were
described
(Linskens
collected
by
Eight discharge
same
turning
tubes
time under
the
capillary
a
15
Determinations of
the
in
12
AND
I
recently
acids
After the exposure
to remove
fixed
was
first
solution
main exposure 6 additional small
added and absorbed.
whole tissue
proline
SCHRAUWEN
the
mg/ml). During
the main part of the
were
the
(20
J.
BRITIKOV,
N
were
the
only
determinations
made with
a
15
4 jxg
was
N
(10~
gave
than
analyser
by
a
100%
%.
spectroscopic
for 3
hrs
tube and
nitrogen
was
at
as
it
has
been
the individual amino
method
The
GMBH, Berlin,
15
16
N 2 in
two
hrs
the
at
samples
parallel
determinations
at
in
cooling,
conversion from
within
into
1967).
preheated
After
difference between
Spectrophotometric
Isocommerz
the
completed
3
(Faust
were
500°C.
sealing,
conversion of NH
nitrogen.
1
analyser
and
5 mg CaO and CuO
Torr)
discharge
of total
less
5
molecular
500°C. Control estimations
1969),
collector.
made
) containing
vacuum
ammonium chloride into
containing
I
automatic
an
Schrauwen
fraction
{fig.
into
using
&
were
University
of
Groningen.
516
Acta Bot. Need.
19(4), August
1970
AS
PROLINE
Fig.
1.
A
SOURCE
A. Tube
NITROGEN
adaptor for
and CuO.
were
OF
When
cooled
down
view
does not
room
to
3.
15
RESULTS
AND
Non-isotopic
means
sharply
within the
The
main
from
as
was
all
10”
500°C,
adaptor
was
5
turned
which contain
and the
Torr)
that the
so
CaO
tubes
capillaries
tubes.
tubes takes
place,
while
the
vacuum
found
to
be present in each fraction
the
of
first
only
showed about 3
nitrogen
was
to
and
pecially
in those of them which
further
transformations, i.e.,
atom
15
are
per
cent
in the
branches in
rest
N-L-proline
to
be
present
compounds
was
closely
naturally
and
related
in
analysed
of amination and
glutamic
1970
N-excess of this
23
proline
by
a
part of free
(table 2). This
was
degraded
released.
isotopic nitrogen
products
15
absorbed
found
was
all individual
of free
%
from that absorbed
(48 hrs)
exogenously
19(4), August
{table 1)
analysed (table 2).
proline
and in
portion
Acta Bot. Need.
discharge tubes,
discharge
of the
decreased from about 95
(table 1)
(FAA)
of 8
also be mentioned that
must
tissues and its
N-nitrogen
acids
J sealing
of the
exposure
practically
15
fraction
At
the
respective
automatic analysis
the end
that
proline
compounds
labelled form. It
proline
temperature,
their
of A.
degassing
completed (3 hrs,
was
DISCUSSION
from
N-nitrogen
into
METABOLISM
change.
in all individual
tissues by
(detail)
PLANT
simultaneous
degassing
simultaneously glided
B. Side
IN
to
each
investigated
(table 2).
found in
free amino
transamination, and
proline degradation
aspartic acid,
arginine,
and
es-
and
maybe
517
E.
A.
BRITIKOV, J. SCHRAUWEN
AND
H. F. LINSKENS
15
Table
1.
N-excess
in
different
fractions
of
Chenopodium
tissues.
15
Fractions
N-atoms
per
Free
amino
Insoluble
acids
10.06*
(FAA)
fraction
cent
(IF)
1.93*
Including:
Bound amino
a.
b.
The
other
acids
(BAA)
1.03**
bases
N-compounds (nucleic
0.90***
etc.)
Amino-lipids
0.99
Chlorophyll
0.35
*
estimated
**
***
As
Table
from the
the difference
2.
Amino
spetrometrically.
mass
Calculated
in
Distribution
of
data
15
on
N-content
15
N-excess
15
N in
between
the
amino
and
15
N-excess
for each
BAA.
IF- and BAA-fractions.
acids
(in
atom per
cent).
free
acids/fractions
amino
bound
amino
acids,
BAA
23.0
3.78
proline
glutamic acid
aspartic
acids,
FAA
1.27
acid
0.73
11.72*
arginine
1.15
alanine
1.14
serine
2.35
1.40
glycine
0.73
0.30
2.72
0.49
7.40
0.98
lieu
Thr, Val, Met, Leu,
His
Tyr, Phe, Lys,
GABA,
EtNH
Met for
BAA)
NH
3
*
2
(plus Om,
for
(amides, Try,
Calculated
alanine
as
hydrolysis
cant
have
to
well
as
(table
for
take
amides
2).
15
15
N-content
high
degraded
even
between
arginine
proline
15
failed
quantitative
as
and had
and
sum
15
and
proline
these latter
have
as
the
a
during
amino
acids
content
accompanied
was
acid
glutamic
might
these
really
was
by signifiseemed
to
direct and transient
highest
15
N-enrichment
accumulation in the tissues.
N-excess
as
high
only 5-12%
a
as
into
1.03
(BAA)
atom
account
day (Hellebust
non-isotopic nitrogen also took part
518
and the
acids
respective
analyze
to
aspartic acid,
N. Then,
destruction
significant, especially taking
tissues is
FAA-fraction
diminutionof
(table 5),
Enrichment of the bound amino acids
ones
the whole
ammonia and the
we
after 48 hrs
enrichment with
significant
to
Unfortunately
N. But since
place
intermediate of
without
3
difference in
accumulation of
a
Cys,
FAA.
individually
found
free NH
from the
of all the rest
and
FAA,
,
in
about
was
per
that
cent.
protein
& Bidwell
1/10
that of the free
This may be considered
1964),
turnover
in the
and that
endogenous
protein metabolism during the
Acta
Bot.
Neerl.
plant
exposure.
19(4), August
1970
AS
PROLINB
Table
3.
SOURCE
OF
NITROGEN
in some free
Changes
of fresh
g
Amino
A
IN
PLANT
amino
METABOLISM
content with
acids
(nitrogen-content in jxg/10
exposure
weight).
acids
in 48 hrs
in
72
hrs
Per
cent
differences
proline
116
72
arginine
197
245
82
98
+
15.7
179
185
+
2.2
232
249
+
aspartic
acid
acid
glutamic
nh
3
alanine
46
43
serine
34
35
the
amino
rest
acids:
insufficient
of the
all
total insoluble fraction
AA-analyser.
the
glutamine
considered
are
be
to
1.3
4.3
-
+
1.3
attract
about
being
lost in
rest
half of
a
the
15
N
columns
latter subfraction may represent first of
since
acids,
only
the
{table I),
of this
Nitrogen
of nucleic
nitrogen
+ 19.5
changes.
It is also of interest that bound amino acids
from the
-37.1
aspartic acid,
utilized in
glycine,
and
purine
asparagine
pyrimidine
and
synthesis
(Meister 1965).
A fraction of the
observed in the
And
finally,
clusion
bolism
of
content
Santalum
N-nitrogen
album does
Chenopodium
natural
on
the
proline
do
species
even
also of
was
with
experiments
15
soluble in
compounds
sumably amino-lipids)
(Girl
important
C-L-proline (Britikov
also involved in
not
belong
the
to
its derivatives in
et
al.
proline
role of
had been earlier
et
al.
1966).
chlorophyll synthesis.
with
plants
extravagant
vegetative
as
organs
as
of
a source
nitrogen
a
high
e.g. Citrus
But this circumstance makes
1952).
(pre-
water
as
I4
was
or
and insoluble in
acetone
high isotopic level, just
for
our
plant
or
con-
meta-
convincing.
more
REFERENCES
Bathurst,
N. O.
tung
des
Prolins
E.
Britikov,
Proline
& N.
A.
in
N.
A.,
in the
Fertilization,
—
(1954):
The
der
A.
Musatova
—
N. A.
on
and
Kartoffel.
S.
Musatova,
(1964b);
Musatova
& S. V.
Physiol.
Durzan,
shoots
H.
&
Vég.
D. J. &
of Picea
Acta Bot. Need.
M.
4:
V.
of
J.
Forsch.
In:
North-Holland
of free
Exp.
Bot.
5:
253-256.
das Vorkommen
12:
Vladimirtseva
plants.
Accumulation
Musatova
Ibid.
12:
H. F.
&
M.
A.
Linskens
Publ.
und
die Bedeu-
293-295.
Protsenko
Pollen
(1964a):
Physiology
and
Co.
proline
in
pollen.
Soviet
Plant
Physio-
Vladimirtseva
Maille
(1965):
Transformation of
proline in germination
839-850.
pollen germination and pollen
Duranton,
pollen.
(1954): Über
II: 394-400.
&N.A.
tissues.
pistil
of grass
Landwirtsch.
77-85, Amsterdam,
p.
S. V. Vladimirtseva
pollen
acids
& O. Fischnich
reproductive system
logy (Fiziologya rastend)
—
amino
F. Heilinoer
Breyhan, Th.,
(1962);
tube
(1966);
growth.
Métabolisme
Effect of
Ibid.
de la
13:
proline
and
its
antimetabolites
860-867.
proline
chez
le
topinambour. Ann.
271-294.
F. C.
Steward
(1963):
glauca (Moench)
19(4), August
1970
The
nitrogen
Voss. Plant
metabolism
Physiol.
38, suppl.,
and seasonal
changes
in
vi.
519
E.
A.
BRITIKOV,
J.
SCHRAUWEN
AND
H.
F. LINSKENS
15
Faust,
H. (1967): Probenchemie
molbereich
Girl,
K.
V.,
Proline
für
H.
strains
—
&
J.
F.
&
J.
Need.
A.
Perdrizet,
pommes
&
de
l’infection.
Shvedskaya,
zation
in
Tupÿ
(1969):
and
terre
C.r.
Z.
J.
and
The
provoquées
Publ.
520
Protein turnover
amino
acids
of free
release
the
& A.
par
Sei., Paris,
S.
Metabolism
(Ed.)
& C. S. Vaidyanathan
170:
(London)
cross-pollination.
amino
Macquaire (1963):
differentiation
(1964) :
Linskens
bis Nano-
3: 100-103.
in
(1952) :
579-580.
attached wheat and tobacco
pool
Der
amino
in the
Züchter
acids
of
style
36:
self-incompatible
151-158.
out of the
germinatingpollen.
605-614.
Acad.
M.
Nature
(1964):
The
(1966):
after self-
18;
G.
im Mikro-
1-12.
Pollen
virus
le
of
acids.
Etude
257:
Kruzhilin
of the
physiology (Fiziologya rastend)
TupY,
leaves.
(1965); Biochemistry of
E.
Stickstoffverbindungen
A. N. Radkakrishnan
Kirshnan,
J. Bot. 42;
Schrauwen
Meister,
-markierter
& R. G. S. Bidwell
of Petunia
Acta Bot.
L.
hydroxyproline
Canad.
Linskens,
N
emissionspectrometrische Isotopenanalyse. Isolopenpraxis
Gopa, K.
I.
and
Hellebust, J. A.,
leaves.
die
N. Y.
Press,
du métabolisme
durant les
London,
des feuilles
premiers stages
II.
de
de
3208-3211.
(1966): Changes
in
in biennial
proline
and
content
winter
during
plants.
vernali-
Soviet
piant
748-755.
proline
Physiology
ed. Acad.
de l’anvirolement
growth points
13:
2nd
des variations
in
and
styles
and
pollen tubes
Fertilization, 86-94,
of Nicotiana
Amsterdam,
alata. In
:
H. F.
North-Holland
Co.
Acta Bot.
Neerl.
19(4), August
1970