



Study with the several resources on Docsity
Earn points by helping other students or get them with a premium plan
Prepare for your exams
Study with the several resources on Docsity
Earn points to download
Earn points by helping other students or get them with a premium plan
Community
Ask the community for help and clear up your study doubts
Discover the best universities in your country according to Docsity users
Free resources
Download our free guides on studying techniques, anxiety management strategies, and thesis advice from Docsity tutors
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
Typology: Study notes
1 / 7
This page cannot be seen from the preview
Don't miss anything!
Received for publication March 7, 1983 and in revised form May 10, 1983
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
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.
the endosperm are metabolized and the products are transferred
(1, 2), but^ at^ the^ same^ time^ storage^ proteins^ are^ hydrolyzed^ to
sperm, absorb the sucrose and amino acids and transport them
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-
characterized (16, 34, 35). Although the fate of the storage lipid and storage protein in
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
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-
which these components are metabolized and transferred to the
these metabolites.
Materials. (^) [8-'4C]Adenine (56 mCi/mmol), (^) [8-'4C]adenosine
phate (51 mCi/mmol), [U-'4C]sorbitol (250^ mCi/mmol),^ [8-3H]
ine hydrochloride (54.7 mCi/mmol), (^) [8-'4C]hypoxanthine (46.
(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
Hale) were soaked for 24 h in cold running tap water, then placed
370
tides in the^ endosperm, the^ procedure described^ by Munro and
ice-cold 0.6 N HCl04 and incubation at 0C for 10 min. After
in 0.1 N KOH.
(2.5-15 (^) Ag/ml), 1 ml^ of 0.04% (w/v) indole^ solution, and^1 ml
mate of the total cellular nucleotide^ content, the^ acid-soluble
A232 ratios^ were 1.0 to^ 0.7 for^ extracts^ from^ 3-^ to^ 9-d-old beans
An extinction of 1.000 at 260 nm corresponded to an AMP
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.
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-
(^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.
from the incubation and analyzed for metabolites.
was determined with ninhydrin following the method of Lee and
from Bergmeyer (3) as follows. Sucrose was measured with
glucose with invertase. Adenosine was determined with adeno-
nucleoside (^) phosphorylase, guanase, and xanthine oxidase. Ad-
mulation of RNA, which reaches its maximal concentration at
who measured the RNA content up to 5 d. With an averaged
proportions of adenine, guanine, cytosine, and uracil, the maxi-
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
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
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
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
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,
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
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
a Km =^ 0.123 mM for adenosine uptake. These values compare with (^) Vm,, for sucrose and glutamine uptake of 1 3 and 69 (^) Amol
Vmax/Km) is of the same order as that of sucrose and glutamine uptake, while that^ of^ adenosine uptake is^ considerably^ higher
space between endosperm and cotyledons in the intact seedling are not known, but the uptake capacities of the cotyledons for
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-
comparison. The other free bases showed considerably lower
hand display uptake characteristics resembling closely those
those of all other compounds. ATP was not taken up by cotyle- dons but interestingly a considerable uptake of AMP occurred
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
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
Glutamine (^) 57.6 (69) 11.8 (11.8) 4. *Value from Ref. 16.
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.
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
molecules (urea, glyoxylate, alanine, C02, NH3, the end products
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
intermediary metabolism, therefore appear to be ideal com-
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.
due to (^) nonspecific hydrolysis of nucleotides released from dam-
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
accommodated by the uptake systems in the (^) cotyledons. The
in the intagt seedling, where endosperm and (^) cotyledons are in
the small space between them might limit the rates of (^) their
(^374) 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'-