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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
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- 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-
