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Transport of Nucleic Acids in Germinating Castor Bean Seedlings, Study notes of Literature

A study on the transport of nucleic acids and nucleosides from the endosperm to cotyledons in germinating castor bean seedlings. The researchers found that RNA is the dominant species in the endosperm and undergoes a massive accumulation during the early stages of germination, which declines to nearly zero by the eighth day. The study also reveals that the rates of metabolite release from the endosperm and the capacity of the absorption system in the cotyledons account for the observed rates of disappearance of nucleotides from the endosperm and efficient transport to the growing embryo. The researchers suggest that nucleosides are the likely transport metabolites between endosperm and cotyledons.

What you will learn

  • What are the likely transport metabolites between endosperm and cotyledons?
  • What is the dominant nucleic acid species in the endosperm during germination?

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Plant
Physiol.
(1983)
73,
370-376
0032-0889/83/73/0370/07/$00.50/0
Transport
of
Purine
and
Pyrimidine
Bases
and
Nucleosides
from
Endosperm
to
Cotyledons
in
Germinating
Castor
Bean
Seedlings'
Received
for
publication
March
7,
1983
and
in
revised
form
May
10,
1983
ERICH
KOMBRINK
AND
HARRY
BEEVERS
Biology
Department,
University
of
California,
Santa
Cruz,
California
95060
ABSTRACI
During
germination
and
early
growth
of
castor
bean
(Ricinus
com-
munis)
all
cellular
constituents
of
the
endosperm
are
eventually
trans-
ferred
to
the
growing
embryo.
The
present
results
bear
on
the
transport
of
breakdown
products
of
nucleic
acids.
The
total
content
of
nucleic
acids
and
nucleotides
declines
rapidly
between
day
4
and
day
8
of
seedling
development.
Concomitant
with
this
decline,
a
secretion
of
adenosine,
gunosine,
and
adenine
from
excised
endosperms
into
the
incubation
medium
takes
place,
accompanying
a
much
more
extensive
release
of
sucrose
and
amino
acids.
Release
of
nucleotides
could
not
be
detected.
The
rates
of
release
were
linear
for
at
least
5
hours
for
all
compounds
measured,
indicating
that
they
were
liberated
due
to
a
coordinated
me-
tabolism.
Uptake
studies
with
cotyledons
removed
from
the
seedling
showed
that
these
have
the
ability
to
absorb
all
the
substances
released
from
the
endosperm.
Besides
sucrose
and
amino
acids,
both
nucleosides
and
free
purine
and
pyrimidine
bases
were
taken
up
by
the
cotyledons
with
high
efficiency.
AMP
was
also
transported
whereas
ATP
was
not.
Kinetic
analyses
were
carried
out
to
estimate
the
maximal
uptake
capac-
ities
of
the
cotyledons.
Rates
of
uptake
were
linear
for
at
least
1
to
2
hours
and
saturation
kinetics
were
observed
for
all
substances
investi-
gated.
It
is
concluded
that
nucleosides
can
serve
best
as
transport
metabolites
of
nucleic
acids,
inasmuch
as
they
are
taken
up
by
the
cotyledons
with
the
highest
efficiency,
the
V./Ka
ratios
being
consid-
erably
higher
than
those
found
for
free
purine
and
pyrimidine
bases.
For
both
adenosine
and
adenine
transport,
the
V,.
was
about
2
micromoles
per
hour
per
gram
fresh
weight,
and
the
K.,
values
were
0.12
and
037
millimolar,
respectively.
The
rates
of
metabolite
release
from
the
endo-
sperm
and
the
capacity
of
the
absorption
system
in
the
cotyledons
are
shown
to
account
for
the
observed
rates
of
disappearance
of
nucleic
acids
from
the
endosperm
and
efficient
transport
to
the
growing
embryo.
During
germination
of
the
castor
bean,
the
storage
reserves
of
the
endosperm
are
metabolized
and
the
products
are
transferred
to
the
growing
embryo.
The
metabolism
in
the
endosperm
is
dominated
by
the
massive
conversion
of
storage
lipids
to
sucrose
(1,
2),
but
at
the
same
time
storage
proteins
are
hydrolyzed
to
amino
acids
(38).
The
cotyledons,
enclosed
within
the
endo-
sperm,
absorb
the
sucrose
and
amino
acids
and
transport
them
to
the
growing
parts
of
the
seedling.
Kriedemann
and
Beevers
(17)
and
Robinson
and
Beevers
(34)
have
demonstrated
that
excised
cotyledons
retain
the
ability
to
absorb
sucrose
and
amino
acids
at
high
rates.
Both
substances
are
accumulated
against
a
I
Supported
by
a
fellowship
of
the
North
Atlantic
Treaty
Organization
Science
Council
via
the
Deutscher
Akademischer
Austauschdienst
to
E.
K.
and
United
States
National
Science
Foundation
Grant
PCM-78-
19575.
concentration
gradient,
and
their
transport
systems
have
been
characterized
(16,
34,
35).
Although
the
fate
of
the
storage
lipid
and
storage
protein
in
germinating
castor
bean
is
clear,
it
remains
to
be
established
what
happens
to
the
other
cell
constituents
of
the
endosperm.
After
8
to
10
d
of
germination,
the
endosperm
has
completely
disappeared
and
only
a
dry,
thin,
papery
skin
from
the
inner
testa
remains,
surrounding
the
cotyledons.
Thus,
virtually
all
the
endosperm
constituents
are
ultimately
transferred
to
the
cotyle-
dons
but
it
is
not
known
in
which
form
and
by
what
mechanism
this
is
done.
Besides
storage
lipid,
protein,
and
intermediates
of
their
pri-
mary
metabolism,
nucleic
acids
(RNA,
DNA),
nucleotides,
and
membrane
phospholipids
are
quantitatively
important
organic
components
of
the
endosperm
tissue.
In
considering
the
way
in
which
these
components
are
metabolized
and
transferred
to
the
cotyledons,
an
interesting
question
is
the
degree
to
which
struc-
tures
requiring
extensive
energetic
expenditure
are
conserved.
The
possible
transport
metabolites
for
nucleic
acids
are
(a)
the
free
purine
and
pyrimidine
bases
(besides
ribose
and
phosphate)
or
(b)
the
nucleosides,
whereas
(c)
the
transport
of
nucleotides
across
membranes
appears
unlikely.
Transport
systems
for
purines,
pyrimidines,
and
nucleosides
have
been
demonstrated
and
characterized
in
bacteria
(5,
13,
28),
yeast
(20,
30,
31),
fungi
(6,
22),
and
animal
cells
(4,
29),
whereas
only
little
information
is
available
about
similar
trans-
port
systems
in
plants
(9,
14,
25,
41).
In
this
paper,
we
describe
the
analysis
of
transport
metabolites
for
nucleic
acids
between
the
endosperm
and
cotyledons
of
the
germinating
castor
bean
and
briefly
report
the
uptake
capacities
of
the
cotyledons
for
these
metabolites.
MATERIALS
AND
METHODS
Materials.
[8-'4C]Adenine
(56
mCi/mmol),
[8-'4C]adenosine
5
'-monophosphate
(43
mCi/mmol),
[8-'4C]adenosine
5'-triphos-
phate
(51
mCi/mmol),
[U-'4C]sorbitol
(250
mCi/mmol),
[8-3H]
guanosine
(17
Ci/mmol),
[5,3-3Hluridine
(40
Ci/mmol),
[5-3H]
cytidine
(21
Ci/mmol),
were
purchased
from
ICN.
[8-'4C]Guan-
ine
hydrochloride
(54.7
mCi/mmol),
[8-'4C]hypoxanthine
(46.0
mCi/mmol),
[2-'4C]uracil
(55.2
mCi/mmol),
[8-'4C]adenosine
(45.2
mCi/mmol)
were
supplied
by
New
England
Nuclear,
and
[2-`4C]cytosine
(9.0
mCi/mmol)
by
DHOM
Products
(Rosechem
Products,
Los
Angeles,
CA).
The
unlabeled
counterparts
of
the
chemicals
listed
above
were
from
Sigma
Chemical
Co.,
as
were
adenosine
deaminase
(from
calf
intestinal
mucosa)
and
guanase
(from
rabbit
liver).
Xanthine
oxidase
(from
cow
milk),
nucleoside
phosphorylase
(from
calf
spleen),
and
all
other
auxiliary
enzymes
and
co-substrates
used
were
from
Boehringer.
Growth
Conditions.
Castor
bean
seeds
(Ricinus
communes
cv
Hale)
were
soaked
for
24
h
in
cold
running
tap
water,
then
placed
in
moist
vermiculite
and
germinated
in
the
dark
at
30°C
in
a
humidified
growth
chamber.
The
time
of
planting
was
taken
as
370
pf3
pf4
pf5

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Download Transport of Nucleic Acids in Germinating Castor Bean Seedlings and more Study notes Literature in PDF only on Docsity!

Plant Physiol. (1983) 73, 370-

Transport of Purine and Pyrimidine^ Bases and^ Nucleosides from

Endosperm to^ Cotyledons^ in^ Germinating^ Castor^ Bean^ Seedlings'

Received for publication March 7, 1983 and in revised form May 10, 1983

ERICH KOMBRINK AND HARRY BEEVERS

Biology Department,^ University^ ofCalifornia,^ Santa^ Cruz,^ California^95060

ABSTRACI

During germination and early growth of castor bean^ (Ricinus com- munis) all cellular constituents of the endosperm are eventually trans- ferred to the growing embryo. The present results bear on the transport of breakdown products of nucleic^ acids.^ The total^ content^ of nucleic^ acids and nucleotides declines rapidly between day 4 and^ day 8 of^ seedling development. Concomitant with this decline, a secretion of adenosine, gunosine, and adenine from excised (^) endosperms into the incubation medium takes (^) place, accompanying a much more extensive release of sucrose and amino acids. Release of nucleotides could^ not^ be^ detected. The rates of release were linear for at least 5 hours for all compounds measured, indicating that they were liberated due to a coordinated me- tabolism. Uptake studies^ with^ cotyledons removed from the^ seedling showed that these have the^ ability to^ absorb all^ the substances released from the endosperm. Besides sucrose^ and^ amino^ acids,^ both^ nucleosides and free purine and pyrimidine bases were taken up by the cotyledons with high efficiency. AMP was also transported whereas ATP^ was not. Kinetic analyses were carried out to estimate the maximal uptake capac- ities of^ the^ cotyledons. Rates^ of uptake^ were^ linear^ for^ at^ least^1 to^2 hours and saturation kinetics were^ observed^ for^ all substances^ investi- gated. It is concluded that nucleosides can serve^ best^ as^ transport metabolites of nucleic acids, inasmuch as they are taken up by the

cotyledons with^ the^ highest efficiency,^ the^ V./Ka ratios^ being^ consid-

erably higher^ than^ those^ found for^ free^ purine^ and^ pyrimidine^ bases. For both adenosine and^ adenine transport, the^ V,.^ was^ about^2 micromoles per hour per gram fresh weight, and the^ K., values^ were^ 0.12^ and^037 millimolar, respectively. The rates of^ metabolite release^ from^ the^ endo- sperm and the capacity of the absorption system in^ the cotyledons are shown to account for the observed rates of disappearance of nucleic acids from the endosperm and efficient transport to the growing embryo.

During germination of the castor bean, the storage reserves of

the endosperm are metabolized and the products are transferred

to the growing embryo. The metabolism in the^ endosperm is

dominated by the^ massive conversion^ of^ storage lipids to^ sucrose

(1, 2), but^ at^ the^ same^ time^ storage^ proteins^ are^ hydrolyzed^ to

amino acids (38). The cotyledons, enclosed within the endo-

sperm, absorb the sucrose and amino acids and transport them

to the growing parts of the seedling. Kriedemann and Beevers

(17) and Robinson and Beevers (34) have demonstrated that

excised cotyledons retain^ the^ ability to^ absorb^ sucrose^ and^ amino

acids at^ high rates.^ Both substances^ are^ accumulated^ against^ a

I (^) Supported by a fellowship ofthe North Atlantic Treaty Organization Science Council via the Deutscher Akademischer^ Austauschdienst^ to^ E. K. and United States National Science Foundation^ Grant^ PCM-78-

concentration gradient, and their transport systems have been

characterized (16, 34, 35). Although the fate of the storage lipid and storage protein in

germinating castor^ bean^ is^ clear,^ it remains^ to^ be^ established

what happens to the other cell constituents of the endosperm. After 8 to 10 d of germination, the^ endosperm^ has^ completely disappeared and only a dry, thin, papery skin from the inner

testa remains, surrounding the cotyledons. Thus, virtually all the

endosperm constituents^ are^ ultimately^ transferred^ to^ the^ cotyle-

dons but it is^ not^ known^ in^ which form^ and by^ what^ mechanism this is done. Besides storage lipid, protein, and intermediates of their pri-

mary metabolism, nucleic acids (RNA, DNA), nucleotides, and

membrane phospholipids are quantitatively important^ organic

components of the endosperm tissue. In considering the way in

which these components are metabolized and transferred to the

cotyledons, an interesting question is the degree to which struc-

tures requiring extensive energetic expenditure are conserved.

The possible transport metabolites for nucleic acids are (a) the

free purine and pyrimidine bases (besides ribose and phosphate)

or (b) the nucleosides, whereas (c) the transport of nucleotides

across membranes appears unlikely.

Transport systems for purines, pyrimidines, and nucleosides

have been demonstrated and characterized in bacteria (5, 13,

28), yeast (20, 30, 31), fungi (6, 22), and animal cells (4, 29),

whereas only little information is available^ about^ similar^ trans-

port systems in plants (9, 14, 25, 41). In this paper, we describe

the analysis of transport metabolites for nucleic acids between

the endosperm and cotyledons of the germinating castor bean

and briefly report the uptake capacities of the cotyledons for

these metabolites.

MATERIALS AND METHODS

Materials. (^) [8-'4C]Adenine (56 mCi/mmol), (^) [8-'4C]adenosine

5 '-monophosphate (43 mCi/mmol), [8-'4C]adenosine 5'-triphos-

phate (51 mCi/mmol), [U-'4C]sorbitol (250^ mCi/mmol),^ [8-3H]

guanosine (17 Ci/mmol), [5,3-3Hluridine (40 Ci/mmol), [5-3H]

cytidine (21 Ci/mmol), were^ purchased from^ ICN.^ [8-'4C]Guan-

ine hydrochloride (54.7 mCi/mmol), (^) [8-'4C]hypoxanthine (46.

mCi/mmol), [2-'4C]uracil (55.2 mCi/mmol), [8-'4C]adenosine

(45.2 mCi/mmol) were supplied by New England Nuclear, and [2-`4C]cytosine (9.0^ mCi/mmol)^ by^ DHOM Products^ (Rosechem Products, Los^ Angeles, CA).^ The unlabeled^ counterparts^ of^ the

chemicals listed above were from Sigma Chemical Co., as were

adenosine deaminase (from calf intestinal mucosa) and guanase

(from rabbit liver). Xanthine oxidase (from cow milk), nucleoside

phosphorylase (from calf^ spleen), and all^ other^ auxiliary enzymes

and co-substrates used^ were^ from^ Boehringer.

Growth Conditions. Castor bean seeds (Ricinus communes cv

Hale) were soaked for 24 h in cold running tap water, then placed

in moist vermiculite and germinated in the dark at 30°C in^ a

humidified growth chamber. The^ time^ of^ planting was^ taken^ as

370

NUCLEIC ACID METABOLITE TRANSPORT IN RICINUS SEEDLINGS

time zero in developmental studies.

Extraction and Separation of Nucleic Acids. For the determi-

nation of the total contents of RNA, DNA, and soluble nucleo-

tides in the^ endosperm, the^ procedure described^ by Munro and

Fleck (23) was followed with slight modifications.

Sixteen seedlings were selected for uniformity at the various

developmental stages and the^ endosperm tissue^ was^ excised.

Endosperm halves^ were^ homogenized in 80^ ml^ ice-cold^ distilled

H20 (VirTis homogenizer). The total^ RNA, DNA, and^ protein

in 5 ml homogenate was precipitated by addition of 2.5 ml^ of

ice-cold 0.6 N HCl04 and incubation at 0C for 10 min. After

centrifugation (15 min, 16,000g), the^ precipitate was^ washed

twice with 5 ml^ cold 0.2^ N^ HC104. The supernatant (acid-soluble

fraction) and the first washings were combined and analyzed for

nucleotides as described below.

For separating RNA and DNA, 4 ml^ of 0.3^ N^ KOH^ were

added to the HC104 precipitate and after thorough mixing the

solution was^ kept at^ 40C^ for^ 1 h^ and then cooled^ in^ ice.^ DNA

and protein were precipitated by adding 2.5 ml of 1.2^ N^ HCl

and, after standing in the cold for 10 min and centrifugation ( 15

min, 16,000g),^ the supernatant^ (RNA^ fraction)^ was^ decanted.

The precipitate was washed twice with 5 ml 0.2 N HCl04 (0C)

and the washings added^ to^ the^ RNA^ fraction.^ Following the

addition of 6 ml 0.2 N HCl04, this fraction was^ made up to^50

ml with water giving a solution ofribonucleotides in^ 0.1 N^ HC104.

To determine DNA, the precipitate obtained on acidifying the

alkaline digest was dissolved in 4 ml 0.3 N KOH by warming

briefly to 400C. The solution was then made up to 25 ml,

including a further 4.3 ml 0.3 N KOH, to give a solution of DNA

in 0.1 N KOH.

RNA Estimation. RNA content was determined spectropho-

tometrically assuming that^ an^ extinction of 1.000^ at^260 nm^ is

equivalent to 48 gg RNA/ml. This^ figure was^ determined^ by

preparing a calibration curve with purified yeast RNA^ (Sigma,

type XI) after^ hydrolysis of^ the^ RNA^ in^ 0.3^ N^ KOH for^1 h^ at

400C and^ measuring the absorbance^ of the^ resulting nucleotide

solution in 0.1 N^ HC104. RNA^ concentrations^ were^ corrected^ for

small protein contaminations in^ different fractions^ as^ described

(10, 23) after measurement of the protein concentration (21).

DNA Estimation. DNA was estimated by reaction with indole

according to^ Ceriotti^ (8). Two ml^ of^ DNA^ solution^ in^ 0.1^ N^ KOH

(2.5-15 (^) Ag/ml), 1 ml^ of 0.04% (w/v) indole^ solution, and^1 ml

concentrated HCl were carefully mixed and incubated in boiling

water for 10 min. After cooling, the solution was extracted three

times with 4 ml CHCl3, centrifuged, and the^ absorbance^ of^ the

water phase read at 490 nm. A standard curve was prepared

using highly polymerized DNA^ from^ salmon^ (Sigma, type III).

Estimation of Total^ Soluble^ Nucleotides.^ To^ get a^ rough esti-

mate of the total cellular nucleotide^ content, the^ acid-soluble

fraction of the endosperm extract (12.5 ml) was adjusted to pH

8 with 1.0 N KOH at 0°C and the precipitate removed by

centrifugation (15 min, 30,000g). The^ supernatant was^ passed

through an^ anion^ exchange column^ (1 x^ 6 cm, Dowex^ 1-X2,

200-400 mesh) and^ washed^ with^12 ml^ 0.01^ N^ NaCl.^ Bound

material was eluted with 20 ml of a solution containing 1.0 M

HCl and 0.4 M NaCl. The eluted solution was made up to 25 ml

and the absorbance was measured at 260 and 232 nm. The A260/

A232 ratios^ were 1.0 to^ 0.7 for^ extracts^ from^ 3-^ to^ 9-d-old beans

and 0.6 to 0.5 for younger beans and dry seeds. The nucleotide

concentration was determined according to Fleck and Begg (10)

from a calibration curve prepared with AMP (Sigma, type II).

An extinction of 1.000 at 260 nm corresponded to an AMP

concentration of 0.073 gmol/ml.

Uptake Experiments with Cotyledons. These were performed

essentially as^ described by Robinson^ and Beevers^ (34). Seedlings

of similar size were selected and the (^) endosperm and (^) hypocotyl were removed. The excised cotyledons (50-55 mg/pair) were

weighed, then transferred to ice-cold buffer (5 mM KH2PO4, 0.

mM CaCl2, pH 6.0) for 20 to 40 min. The cotyledons were then

lightly blotted and placed in a solution of the same buffer

containing the desired compound for uptake measurement (1 ml

buffer per cotyledon pair). After incubation at 25°C for 5 min,

0.5 to 1.0 (^) ,gCi of the radioactive-labeled compound was added and the incubation was continued in the dark in a shaking water bath. Uptake was normally determined from the loss of radio-

activity in the incubation solution. Triplicate samples of 20 to

(^50) ,l were withdrawn at^ time intervals of 15 to 30 min for 1 to 3 h and uptake rates were calculated from the time curve so obtained. Radioactivity was determined by liquid scintillation counting.

One volume sample plus water was added to 10 volumes of

scintillation mixture which was toluene:Triton X-100 (2:1, v/v)

plus 0.4% (w/v) 2,5-diphenyloxazole and 0.02% (w/v) p-bis-[2-

(5-phenyloxazolyl)]benzene.

Release of Metabolites from Endosperms. To identify possible

transport metabolites and measure their rates of release, endo-

sperm halves were incubated in buffer (5 mm KH2PO4, 0.1 mM

CaCl2, pH 6.0) at^ 250C in^ a^ shaking water bath, after they had

been washed briefly (5 min) in^ the same buffer. Normally five

endosperm halves were placed in a 100-ml beaker and were just

covered with buffer, usually 7.5 ml. Alternatively, to avoid

possible 02 limitation in the incubation, 15 ml buffer and 10

endosperm halves were placed in a 30-ml fritted glass funnel

(Pyrex No. 36060, ASTM 4-5.5 F) and flushed with compressed

air from below. At 1-h^ time intervals, samples were removed

from the incubation and analyzed for metabolites.

Analytical Methods. Soluble amino nitrogen (amino acids)

was determined with ninhydrin following the method of Lee and

Takahashi (19), L-glycine was used as a standard. All other

metabolites were analyzed by standard enzymic methods adopted

from Bergmeyer (3) as follows. Sucrose was measured with

hexokinase and glucose 6-P dehydrogenase after hydrolysis to

glucose with invertase. Adenosine was determined with adeno-

sine deaminase, inosine and guanosine were measured using

nucleoside (^) phosphorylase, guanase, and xanthine oxidase. Ad-

enine and guanine were deaminated with HNO2 and guanase,

respectively, and the resulting hypoxanthine and xanthine meas-

ured using xanthine oxidase. Control experiments were per-

formed to ensure that the adenine assay was not distorted by the

presence of 0.1 to 1.0 mM adenosine. ATP was measured with

hexokinase and glucose 6-P dehydrogenase, ADP and AMP with

pyruvate kinase, lactate dehydrogenase, and myokinase. Protein

was determined after TCA precipitation by the method of Lowry

et al. (21) using BSA as standard.

RESULTS

Changes of Nucleic Acids^ and^ Nucleotides during Develop-

ment. An^ analysis of^ the^ major purine and pyrimidine compo-

nents present in^ the^ endosperm of the germinating castor bean

showed that RNA is the dominant species (Fig. 1). The early

stage of germination (1-3 d) is characterized by a massive accu-

mulation of RNA, which reaches its maximal concentration at

4 d. In the following days, the RNA content declines again to a

value of nearly zero at 8 d. The developmental change of RNA

agrees closely with^ the^ previous results^ of Roberts^ and^ Lord^ (33)

who measured the RNA content up to 5 d. With an averaged

mol wt of 321 for nucleotides in RNA and assuming equal

proportions of adenine, guanine, cytosine, and uracil, the maxi-

mal value of 1.2 mg RNA/endosperm is equivalent to 3.74 ,umol

nucleotides (or free bases) per endosperm bound in RNA. The DNA content, on the other hand, is constant during the first 4 d of development and it is much lower than the maximal RNA (^) content. The concentration of about 0.17 mg/endosperm

(equivalent to^ 0.55^ gmol deoxynucleotides/endosperm) declines

NUCLEIC ACID METABOLITE TRANSPORT IN RICINUS SEEDLINGS

Table I. Release ofCompoundsfrom Excised Endosperm Five endosperm halves of 5-d-old seedlings were^ incubated^ in^ 7.5^ ml buffer (5 mM KH2PO4, 0.1 mM CaCl2, pH 6.0) in a shaking water bath at 25C. Substances released were measured after a 2-h incubation. (Also listed in this table are the maximal uptake capacities of cotyledons as measured in Table (^) II, for comparison.)

Rate of Release Uptake Rate of Substance in Buffer + Cotyledons in Buffer

0.4 M^ sorbitol (V^ .=)

,gmol h-'^ endosperm'^ ;Lmol h-'

seedling-' Sucrose 24.0 7.0 6. Amino acids 12.6^ 3.4^ 3. Adenine 0.009 0. Adenosine 0.050 0.008 0. AMP _.a 0. ADP - NDb ATP -^ - Guanine 0. Guanosine 0.026 0.006 0. Inosine 0.003^ 0.001^ ND

, not detectable. ND, not^ determined.

1'.5-

a1.

0 30 60 90 120 150 Incubation Tire(min) FIG. 4. Uptake of adenosine (0) and^ adenine^ (0) by excised^ cotyle- dons. Cotyledons of five seedlings were incubated in^5 ml^ buffer^ (5 mm KH2PO4, 0.1 mm CaCI2, pH 6.0) containing 0.2 mM adenosine (0.5 Ci/ mol) or 0.2 mm adenine (1.0 Ci/mol), respectively. Triplicate samples of

20 gl were removed at intervals and uptake was calculated from the

decrease in^ radioactivity.

amount and rate of release was altered by these incubation conditions. The decrease of the rate of sucrose^ release^ during the^ incuba- tion (Fig. 2) suggested that the increasing solute concentration (osmolarity) in the incubation medium might influence the release rates. When increasing concentrations of sorbitol were included in^ the^ incubation^ medium, significantly^ lower^ rates^ of release from the^ endosperm were^ measured^ for^ all^ metabolites (Fig. 3). In^ 0.4^ M^ sorbitol, the^ rates^ were^ reduced^ to^ about^ one- fourth of the (^) original values (^) (Table I). The release of all com- pounds was^ linear^ for^ at^ least^5 h^ (in Fig.^2 shown^ for^ sucrose and amino acids only). It is clear that the rates of release of^ sucrose^ and^ amino^ acids from the endosperm into^ the^ medium^ not^ containing sorbitol considerably exceed^ the^ maximal^ uptake capacities^ of^ the coty- ledons for these compounds (16, 34). However, with 0.4 M sorbitol in the incubation medium, the rates of sucrose and amino acid release from^ the^ endosperm are^ within the^ same range as the uptake capacities of the^ cotyledons (as^ judged^ by

the V.,,,) for these compounds (Table^ I).^ It is tempting to suppose

that the latter condition more closely resembles that in vivo, with sucrose in the space between endosperm and cotyledons limiting the rate of loss from^ the^ endosperm. Uptake Studies with Excised Cotyledons. From the previous experiments, it seems that nucleosides (and possibly also the free purine and pyrimidine bases) are the likely transport metabolites between endosperm and cotyledons.^ Therefore,^ the^ ability^ of cotyledons to absorb those compounds secreted by the endo- sperm was investigated further, using 4- to 5-d-old cotyledons. When cotyledons were incubated with radioactive substrates, uptake could be determined by measuring^ the decrease^ of^ radio- activity from the external solution, as has been described previ- ously (34). Uptake ofadenine and adenosine was linear for about 1 to 2 h (Fig. 4). The rates of uptake by cotyledons were not significantly changed when the hypocotyl was removed, as^ has been demonstrated for sucrose and glutamine uptake (16, 34); hence, all further experiments were done with isolated cotyle- dons. Kinetic analyses were carried out to estimate the maximal uptake capacities of^ the^ cotyledons. The^ rates^ of^ adenine and adenosine uptake by cotyledons as functions of the^ substrate concentrations are shown in Figure 5. The uptake displayed saturation kinetics with maximal^ rates occurring at^ concentra- tions above 1 to 2 mM. At high substrate concentrations, uptake rates were calculated from several successive measurements in time periods of up to 2 h (cf Fig. 4), whereas shorter incubation times had to be used^ with^ low^ substrate^ concentrations.^ Further- more, during the uptake of adenosine at the lowest concentra- tions measured (0.020-0.050 mM), the external solution was depleted of the^ substrate^ by up to 35% within only 30^ min,

whereas about 90 min were required for the same amount of

adenine to be taken up at the same concentration. In analyses of the data, this was corrected by using the arithmetical average substrate concentration during the time^ interval^ uptake was

measured (18). The kinetic parameters were obtained by linear

regression of v against v/s (Eadie-Hofstee plot) and resulted in a V,,= 2.24^ ,mol h-'^ g^ fresh^ weight-'^ and^ a^ Km^ =^ 0.37^ mm^ for

adenine uptake and a V,,.= 2.14 ,Amol h-'^ g fresh^ weightr' and

a Km =^ 0.123 mM for adenosine uptake. These values compare with (^) Vm,, for sucrose and glutamine uptake of 1 3 and 69 (^) Amol

h-I g fresh weight-' respectively, reported previously for this

tissue (16, 34). The efficiency of adenine uptake (expressed^ as

Vmax/Km) is of the same order as that of sucrose and glutamine uptake, while that^ of^ adenosine uptake is^ considerably^ higher

(Table II). The concentrations of adenine and^ adenosine^ in the

space between endosperm and cotyledons in the intact seedling are not known, but the uptake capacities of the cotyledons for

these compounds (as judged by their V,,) clearly exceed the

secretion rates from the endosperm (Table I). The low^ Km^ values for uptake by the cotyledons would presumably result in efficient removal of adenine and adenosine from the space between them and the endosperm. The uptake of other purine and pyrimidine bases^ and nucleo-

sides was investigated. They also showed saturable uptake kinet-

ics and the determined V,,,,and Km values are listed in Table II,

together with those found for^ sucrose and^ glutamine^ uptake^ for

comparison. The other free bases showed considerably lower

uptake rates (V,,,,) than adenine and the efficiency of their

uptake (Vmax/Km) is lower, too. The nucleosides on the other

hand display uptake characteristics resembling closely those

found for adenosine, although the V,,. and Km are lower, the

Vmaffi,JKm ratios are nearly the^ same and^ clearly^ different^ from

those of all other compounds. ATP was not taken up by cotyle- dons but interestingly a considerable uptake of AMP occurred

with a V,,, similar to that of adenine and adenosine,^ but with a

higher Km. Sorbitol uptake was measured as a kind of control

374 KOMBRINK AND BEEVERS

I

V

Concentrtion (mM) V/S FIG. 5. Concentration dependency of adenosine and adenine (^) uptake by cotyledons (A). Uptake was (^) measured as described in "Materials and

Methods," with 0.5 (@) and 1.0 MCi (0) labeled substrate added per sample. Panel B shows the Eadie-Hofstee plot of the data, the correlation

coefficients are 0.987 (@) and 0.973 (0), respectively.

Table II. Characteristics (^) ofAbsorption ofMetabolites by Castor Bean Cotyledons Cotyledons of five seedlings (4 d old) were incubated in 5 ml buffer (^) ( mM KH2PO4, 0.1 mM CaCl2, pH 6.0) plus the indicated (^) substrates, as described in "Materials and Methods." Substrate (^) VM= Km (VmgJKm)

iAmol h-'^ mM^ ratio g' fresh wt Adenine 2.24 0.373 6. Guanine 0.136 0.055 2. Hypoxanthine 0.742 (^) 0.454 1. Uracil 0.184 0.041 4. Cytosine 0.508 0.335 1. Adenosine 2.14 0.123 17. Guanosine 0.666 0.058 11. Uridine 0.340 0.0175 (^) 19. Cytidine 0.267 0.0223 12. AMP (^) 2.38 0.885 2. ATP No transport Sorbitol No (^) transport

Sucrose 108 (113r 18 (25)8 6.

Glutamine (^) 57.6 (69) 11.8 (11.8) 4. *Value from Ref. 16.

b Value from Ref. 34.

experiment Less than 1% of the (^) radioactivity disappeared from the incubation solution in 30 min and after that (^) time no further uptake of^ sorbitol^ occurred.^ Presumably, the^ initial loss of sor- bitol from the medium is (^) due to nonspecific movement into the free (^) space of (^) the tissue; the fact that the uptake of other com- pounds continues unchanged for several hours indicates the selectivity and specificity of the (^) transport system.

DISCUSSION

Although RNA and nucleotides are (^) readily detectable in dry seeds of castor beans (32, 33; see also (^) Fig. 1), a massive de (^) novo synthesis of these-cell constituents occurs after germination (^) (Fig. 1). From the developmental changes in the concentrations of these (^) compounds in the (^) endosperm (Fig. 1), it is obvious that total RNA represents the (^) major component ofbound purine and pyrimidine bases, (^) amounting to a maximal concentration (^) at day (^4) equivalent to about 3.74 (^) ,umol/endosperm, whereas soluble nucleotides and DNA amount to only 0.8 and 0.55 (^) ,umol/ endosperm, respectively. We did not analyze the tissue for free purine and (^) pyrimidine bases (^) (or other (^) nonphosphorylated deriv- atives), for it is known that (^) they have (^) only a (^) transitory existence

as metabolic intermediates and only occur in minor concentra-

tions in plants (7). Inasmuch as all the above mentioned com-

pounds decline again to nearly zero concentrations at the late

stage of seedling development, a maximal rate of nucleotide

disappearance from the^ endosperm of 50 to 60 nmol h-' seed-

ling-' at^6 to^8 d^ can^ be calculated. This rate must be accounted

for either by conversion of the breakdown products of nucleic

acids and nucleotides (i.e. purine and pyrimidine bases) to small

molecules (urea, glyoxylate, alanine, C02, NH3, the end products

of purine and pyrimidine catabolism) which can enter interme-

diary metabolism again, or by secretion ofbigger building blocks

such as nucleotides, nucleosides, or free bases, and transporting

them to the embryo, the growing part of the plant.

Nucleotides normally do not pass membranes, unless (^) specific transport systems are present, such as those in chloroplasts and mitochondria. (^) The free purine bases, especially guanine, display

only a limited solubility in neutral aqueous media. Nucleosides,

being readily soluble in water and of only limited importance in

intermediary metabolism, therefore appear to be ideal com-

pounds to serve as transport metabolites. Indeed, in confirmation

ofthis view, significant amounts ofnucleosides (adenosine, guan-

osine) were released (^) into the incubation medium when endo- sperm halves were incubated in buffer (Fig. 2). Significant amounts of adenine also appeared in the incubation (^) medium, while adenosine phosphates and guanine were not detectable.

Although it cannot be excluded that the release of nucleosides is

due to (^) nonspecific hydrolysis of nucleotides released from dam-

aged cells on the surface of the endosperms, the linearity of the

release from an incubation time of at least 5 h (^) indicates that the release is most likely due to a (^) coordinated metabolism. The dominance of adenosine in the incubation medium also is (^) not surprising, inasmuch as in most plant tissues the adenosine nucleotides predominate (^) (7). Sucrose and amino acids are by far the most (^) abundant com- pounds to be transferred from the endosperm to the (^) cotyledons of the germinating castor bean, and the transport (^) systems for sucrose and amino acids in cotyledons of castor (^) bean have been characterized in some detail (16, 34, 35). (^) Interestingly, the release of these compounds from the endosperm occurs, with a much higher rate than the maximal uptake capacities (^) ofthe cotyledons, when endosperms were incubated only in buffer. (^) However, when endosperms were incubated in 0.4 M (^) sorbitol, the amounts of

sucrose and amino acids released from the endosperm could be

accommodated by the uptake systems in the (^) cotyledons. The

latter conditions, presumably, more closely reflect the situation

in the intagt seedling, where endosperm and (^) cotyledons are in

close contact (17) and the solute concentration (osmolarity) in

the small space between them might limit the rates of (^) their

(^374) Plant (^) Physiol. Vol. (^) 73, 1983

376 KOMBRINK AND BEEVERS Plant Physiol. Vol. 73, 1983

3 1. REICHERT U, M WINTER 1974 Uptake and accumulation of purine bases^ by 36. Ross CW 1981^ Biosynthesis of nucleotides. In A^ Marcus, ed, The^ Biochemistry stationary yeast cells pretreated with glucose. Biochim Biophys Acta 356: of Plants, Vol 6. Academic Press, New York,^ pp 169- 108-116 37. SHERWIN JE, SA GORDON 1974 Linear velocity of cyclic adenosine 3',5'-

  1. ROBERTS LM, JM LORD 1979 Ribonucleic acid synthesis in germinating castor monophosphate transport in corn coleoptiles. Plant Physiol 53: 416- bean (^) endosperm. J (^) Exp Bot 30: 739-749 38. STEWART CR, H BEEVERS 1967 Gluconeogenesis from amino acids in germi-
  2. ROBERTS LM, JM LORD 1979 Developmental changes in^ the activity of^ nating castor bean endosperm and its role in transport to the embryo. Plant messngerRNAisoltedfromgermnatng csto bea endspem. Pant Physiol^ 42: 1587- messenger RNA isolated from germinating castor bean endosperm. Plant 39. Suss J, J TuPY 1982 Kinetics of uridine uptake and incorporation into RNA Physiol 64: 630-634 (^) in tobacco pollen culture. Biol Plant (Prague) 24: 72-
  3. ROBINSON SP, H BEEVERS 1981 Amino acid transport in germinating castor 40. (^) SUZUKI T, E (^) TAKAHASHI 1977 Biosynthesis of purine nucleotides and meth- bean seedlings. Plant Physiol 68:^ 560-566^ ylated purines in higher plants. Drug Metab Rev 6: 213-
  4. ROBINSON SP, H BEEVERS 1981 Evidence for amino-acid:proton cotransport 41. WASTERNACK CH 1976 Uptake and incorporation of pyrimidines in Euglena in Ricinus cotyledons. Planta 152: 527-533 gracilis. Arch Microbiol 109:^ 167-