Download Received June 19, 1964.

Comparison of Incorporation and Metabolism of RNA Pyrimidine
Nucleotide Precursors in Leaf Tissues 1. 2
Cleon Ross
Department of Botany and Plant Pathology, Colorado State University, Fort Collins
carbamylase, and demonstrated several of its properties. Buchowicz and Reifer (7) found orotic acid
to be converted to UMP, CMP, uridine, and uracil
in wheat seedling leaves. Carbamyl aspartate also
labelled these compounds and, in addition, orotic acid
(30). This work suggests a pathway of nucleotide
synthesis similar to that in other organisms. However, the latter workers reported that the conversion
of orotic acid to UMP involved uridine as an intermediate product (9), and were unable to detect any
OMP in their wheat leaves. Buchowicz (8) also reported that uracil could be incorporated directly into
the nucleotides of RNA in wheat seedling homogenates without first labelling UMP and CMP. A
direct binding of uracil by a polynucleotide acceptor
was suggested to explain this surprising result.
The present experiments were undertaken to provide more evidence as to some of the reactions involved in synthesis of uridylic and cytidylic acids in
higher plants and to extend the results to plants
other than wheat. Quantitative comparisons of the
rate of incorporation of certain precursors into RNA
were made and several metabolites were identified,
including OMP.
The pathway of synthesis of cytidylic and uridylic
acids has been tentatively established in certain microbial and animal cells according to recent reviews
(14, 35, 28). The sequence of reactions is probably
-as follows: CO, + ATP + NH3 carbamyl
-H^O
aspartate
diphosphate
~ carbamyl aspartate -2 H
PRPP3
hydroorotic acid orotic acid
> oro-tidine-5'-phosphate UMP + CO2. In yeast,
UMP was further converted to UTP by kinases requiring ATP (23). Amination of uridine nucleotides to form cytidine nucleoticles involved participa-tion of glutamine in mammalian cells (18, 34) and
NH3 in Escherichia coli (21).
Neither uracil or uridine, cytosine or cytidine, are
normal intermediates in synthesis of uridylic and cy-tidylic acids. The only pyrimidine involved is orotic
acid, according to this pathway. In higher plants,
uracil, uridine, and cytidine are incorporated into
RNA (2, 8, 15), although incorporation of cytosine
has apparently not been reported. Anabolism of
uracil may occur either by addition of ribose from
ribose-1-phosphate to produce uridine (11) or by
-conversion directly to UMP upon reaction with
PRPP (11, 13). Uridine was converted to UMP in
the presence of ATP and a uridine kinase (11). The
enzymes responsible for cytidine utilization have apparently not been extensively studied.
Considerably less is known of the pathway of pyrimidine nucleotide synthesis in higher plants. In
.a preliminary communication Kapoor and Waygood
(19) reported in wheat embryos the presence of the
-enzyme converting orotic acid to OMP, orotidine-5'-phosphate pyrophosphorylase, and of dihydroortic de-hydrogenase, which forms orotate from dihydroorotate. In a similar brief report, King and Wang
(20) showed that C14 from both uracil and aspartic
acid labelled pyrimidines of wheat leaves. Neumann
an1ld Jones (25) recently studied aspartic acid trans1
Materials and Methods
Plants Used. In most of the experiments bean
plants, Phaseolus vulgaris var. Idaho 111, were used.
Leaf discs (1.2 cm diameter) were cut with a cork
borer from the leaflets of the first trifoliate leaves
when these leaflets were 2.5 to 3.8 cm long. Discs
were also cut from the most rapidly growing leaves
(7-8 cm long) of the cocklebur, Xanthium pennsylvcanicum Wall., for use in some of the studies.
Chemicals. Except for uridine-2-C14 and DLcarbamyl-C14-aspartic acid which were obtained from
the New England Nuclear Corporation, all compounds were of the best grade supplied by the California Corporation for Biochemical Research. All
radioactive compounds were chromatographed 2-dimensionally in the BAW and t-BKW solvents described further below. Significant impurities were
not present in any except perhaps carbamyl aspartic
acid. Here we found other compounds present after
chromatography to the extent of 4.7 % of the total C14.
Unlabelled carbamyl aspartic acid and 8-ureidopropionic acids were synthesized by alkaline treatment
of dihydrouracil and dihydroorotate, respectively.
'Received June 19, 1964.
GB-907 from the National Science
Supported by grant
Foundation.
3 Abbreviations: PRPP, 5-phosplhoribosyl-l-pyrophos-phate; UMP, UDP, UTP are 5'-mono, -di, and -triphosphates of uridine; CMP, CDP, CTP, cytidine nucleotides;
uridylic and cytidylic acids refer to the products of alkaline hydrolysis of RNA; OMP, orotidine-5'-phosphate;
2
UDPG, uridine diphosphate glucose; PUP, 0-ureidopro-
Pionic acid.
65
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66
PLANT PHYSIOLOGY
Methods of Supplying Labelled Compounds to the
Tissues. Unless otherwise noted, 10 leaf discs (about
275 mg fresh weight for cocklebur and 225 mg for
bean tissues) were added to 125 ml Erlenmeyer flasks
.containing 5.0 ml 0.01 M phosphate buffer at pH 5.8.
These flasks had a 1.5 cm diameter by 2.5 cm long
well glued to the inside wall about 3 cm above the
bottom. To this well 1.0 ml 3 % KOH was added
to absorb CO2 released in respiration. Solutions of
the radioactive compounds at equal total and specific
radioactivities were added to the buffer solutions.
Usually about 2.0 ,uc of each were used.
After stoppering the flasks they were kept in
darkness on a constant temperature water bathshaker, usually at 28° for 4 hours.
Fractionation of Radioactivity in the Tissues.
Analysis of C14 in respired CO, was performed as described previously (32).
The leaf discs were washed with water to remove
unabsorbed compounds, then killed in 50 ml hot
80 % ethanol and boiled until chlorophyll free. This
solution was decanted and the discs were rinsed
with enough 80 % ethanol so that the combined volume was 100 ml. Aliquots were plated on stainless
steel planchets and C14 measured. The extract was
combined in a separatory funnel with 2 volumes ethyl
ether, 1 volume water, and 0.5 volume cyclohexane.
After shaking, the lower aqueous phase was chlorophyll free. It was evaporated at room temperature
with air or at 40° in a Rinco rotating evaporator
under reduced pressure. The residue was redissolved
in water for chromatography.
The decolorized leaf discs were next homogenized
in 20 ml of 50 % ethanol (previously adjusted to pH
4 with acetic acid) using an Omni mixer. After
centrifugation and rinsing, aliquots were plated and
C14 determined. The residues were further extracted
twice with cold 0.2 N perchloric acid. The extracts
were neutralized with KOH, chilled to precipitate
KClO4, and aliquots plated to measure radioactivity.
Corrections for self-absorption were required and
made. The remainder of the 50 % ethanol and perchloric acid extracts were evaporated as above,
redissolved in water, and combined with the concentrated 80 % alcohol extracts for chromatography.
RNA was extracted with 0.3 M KOH and measured
spectrophotometrically as described previously (31).
Material insoluble in KOH and that precipitated from
the KOH extracts by adjustment to pH 1 (including
DNA) were analyzed for radioactivity in dry powder
form. Approximate self-absorption corrections were
applied. In a few cases these powders were converted
to CO2 by a wet combustion technique described previously (36). After conversion to BaCO3, radioactivity was measured at infinite thickness as above.
Radioactivity Measurements. These were made
using a Nuclear Chicago Corporation gas flow geiger
tube covered by a micromil window, and connected
to an automatic sample changer. All samples except
the BaCO3 were prepared on identical stainless steel
planchets to insure uniform counting geometry.
Self-absorption corrections were required for all except the ethanol soluble extracts. Counting efficiency
was about 32 %.
Chromatography, Electrophoresis, and Autoradiography. Whatman 3 MM papers, 46 X 57 cm,
were used for chromatography of the combined alcohol
and neutralized perchloric acid extracts. Initial separations were usually made with n-butanol-acetic acidlwater (BAW, 2: 1: 1 v/v) followed by t-butanolmethyl ethyl ketone-water-NH4OH (t-BKW, 4: 3:
2: 1 v/v) (16). For rechromatography, to aid in identification of the compounds thus resolved, the following
additional solvents were employed: isopropanol-formic acid-water (IFW, 7: 1: 2), 95 % ethanol-i at
ammonium acetate (7: 5: 3, pH 3.8 or 7.5, see ref.
26), n-propyl acetate-formic acid-water (11: 5: 3)
and n-propanol-NH4OH-water (6: 3: 1, see ref. 5).
and Pabst solvent III (1). Various 2-dimensional
combinations of these were used as found appropriate.
All were run descendingly.
Paper electrophoresis was performed on 3.8 X 5/7
cm strips of paper, using 0.03 M citrate buffer at pH
3.5 or 5.2. A voltage gradient of about 20 v/cm was
maintained. Strips were immersed in a large bath
of carbon tetrachloride to avoid overheating. C14
on these papers was determined using a Vanguard
Model 800 strip scanner.
Autoradiograms of the 2-dimensional chromatograms were made with Kodak Blue Brand X-ray
film. Exposure periods of 2 to 6 weeks were used.
Spots thus detected were cut out, placed in planchets,
and counted directly. Radioactivity in each metabolite was expressed as a per cent of the total detectable C14 present, uncorrected for absorption by the
paper.
For determination of the specific activities of RNA
cytidylic and uridylic acids, chromatography of the
nucleotides was performed in isopropanol-HCl-water
(170: 41: 39 v/v). Nucleotides were detected with
a Mineralight lamp, eluted with 0.1 N HCO and their
concentrations determined in a Beckman DU spectrophotometer. C14 was measured in these same solutions.
Identification of Metabolites. UDPG was identified as follows. Its ultraviolet absorption spectrum
matched that of commercial UDPG or other uridine
nucleotides. It cochromatographed with commercial
UDPG in 5 solvent systems and the 2 moved together
upon electrophoresis at both pH 3.5 and 5.2. In
IFW and BAW partial hydrolysis occurred and radioactive compounds subsequently chromatographing
with UDP, UMP, and less often, uridine were formed.
By these methods the possibility that the spot contained appreciable amounts of UDP-acetylglucosamine
was eliminated, but the presence of UDP-galactose
or other closely related sugar derivative of UDP is
possible.
UMP was identified by its absorption spectrum in
acid and alkaline solution and by cochromatography
in several solvents. When 2-dimensional cochromatography was performed in conjunction with auto-
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ROSS-PYRIAIIDINE NUCLEOTIDE SYNTHESIS
radiography, the ultraviolet absorbing spot on the
paper (detected with a Mineralight lamp) matched
both in shape and position the exposed area on the
film. UDP was found to cochromatograph with the
known compound 1-dimensionally in 4 solvents and to
have the same movement upon electrophoresis at
pH 3.5. The latter method eliminated the chance
that a significant amount of labelled CDP was present
in the UDP area. UDP was also found to hydrolyze
when chromatographed in acid solvents. Under these
conditions some C14 was detected in a position corresponding to the movement of UMP.
The following compounds were identified only by
2-dimensional chromatography, but in all cases matching of the exposed area on the film with the shape and
position of the known compound on the paper left
little doubt as to their identities: orotic acid, uracil,
uridine, dihydrouracil, ,6-ureidopropionic acid, and
carbamyl aspartic acid. The latter 3 compounds were
located after cochromatography with the knowns by
first opening the rings (if needed) by spraying with
0.5 N NaGH. After the papers dried they were
sprayed with p-dimethylanminobenzaldehyde. Yellow
colors resulted.
The presence of labelled sucrose wvas confirmed
by chromatography and autoradiography as above,
and, in addition, it gave a yellow-green reaction characteristic for sucrose when sprayed and heated with
aniline diphenylanmine (Sigma Chemical Company).
Glucose gave a blue-grey color in this reaction.
Two unknown compounds were believed to be oroti(line and orotidine-5'-phosphate because they contained C14 when the leaves absorbed orotic acilcarboxyl-C14. They were chromatographed 2-dimensionally with synthetic orotidine and OMP and
then autoradiographs were made. Solvent systems
were: A) BAW followed by t-BKW, B) IFW followved by Pabst III, C) BAW followed by the pH
3.8 ethanol-ammonium acetate solvent. In all cases,
coincidence of the exposed area on the films with the
spots on the paper detected with the Mineralight lamp
appeared to confirm identities. Finally they were
subjected to electrophoresis with commercial orotidine
and OMP. The movement of C14 exactly matched
the movement of the known compounds.
Results and Discussion
Coniparison of Incorporation of RNA Precursors.
The distribution of C14 in various bean leaf tissue
fractions after addition of several pyrimidine nucleotide precursors is shown in table I. The results sunmmarize 6 separate experiments in which 2 to 4 labelled
precursors were studied at a time under the same
experimental conditions. About 7.8 % of the added
cytosine was absorbed. Absorption was less for
aspartic acid, uridine, uracil, and orotic acid (5.74.2 % of that added), while only 1.9 % of the
carbamyl aspartic acid was taken up. For all compounds, the greatest recovery of C14 was in the combined ethanol extracts. \With the exception of cyto-
67
sine all the compounds were extensively metabolized,
and incorporation into RNA was easily measurable.
Only traces of C14 were found in CO., and in RNA
after feeding cytosine-2-C14, and amounts in RNA
were too low to determine whether cytidylic or uridylic acid, or both, contained the label. This result
supports those of others who found cytosine to be
poorly converted to RNA of rat tissues (4). The
present data also show that the poor anabolism is
not due to a rapid breakdown to CO,.
The label from DL-aspartic acid-3-C14 was incorporated into the RNA in relatively low amounts. Most
of it was recovered in the aqueous ethanol extracts
and in CO.,. Chromatograms of the nucleotides resulting from KOH hydrolysis of the RNA showed
that the majority of the C14 was present in uridylic
acid. Amounts in cytidylic acid were not detectable
by our methods. However, DL-carbamyl-C'4-aspartic
acid was a more efficient RNA precursor than aspartic
acid and was far less rapidly catabolized to CO.,
Only the pyrimidine nucleotides of RNA were detectably labelled; the specific activity ratio of cytidylic
acid/uridylic acid (C/U) averaged 0.63. Reifer
et al. (30) found L-carbamyl aspartic-C14-acid to be
incorporated into these nucleotides in excised wheat
leaves and had previously concluded (29) that it was
metabolized in plants along the same path as in
animals.
Orotic acid-2-C14 was very efficiently utilized in
RNA synthesis and, like carbaryl aspartate, was no,
rapidly catabolized to CO.}. Cytidylic and uridylic
acids contained all the detectable C14 in RNA and the
C/U ratio averaged 0.60. Conversion of uracil-2-C'4
to RNA was approximately half as efficient as orotic
acid conversion. Both cytidylic and uridylic acids
were labelled. Part of this relatively poor utilization
of uracil for RNA was apparently due to competing
catabolic reactions, since one-third of the C14 absorbed
in this compound was recovered as CO.,. Uridine-2C14 was catabolized to a greater extent than either
carbamyl aspartate or orotate, but only about half as
rapidly as uracil, based on radioactivity of the CO..
Incorporation of uridine to RNA was very extensive,
nearly equal to that of orotic acid. This suggests the
adequate presence of a kinase to convert uridine to
UMP.
Other workers who studied both plants (15) anI
animals (10) have found a rapid catabolism of uracil.
Canellakis (10) showed that the relative effectiveness
of uracil, orotic acid, and UMP depended on the
amounts added. At high levels of each, uracil was
almost as efficiently used as the others, while the percentage of uracil breakdown was much less than at low
substrate levels. Thus, our results in table I might be
expected to vary if different precursor concentrations
were provided. However, we have found almost
identical distribution of C14 among various tissue
fractions when cytosine, uracil, and aspartic and orotic
acids were supplied at one-fifth the amounts of table
I, but have no data for larger differences in concentrations of added substrates.
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'co
PLANT PHYSIOLOGY
Table I. Distribution of C14 from Suspected RNA Precursors Among Tissue Fractions of Bean Leaf Discs
Ten 1.2 cm diameter leaf discs (225 mg fresh tissue) were incubated in 5.0 ml 0.01 x potassium phosphate (pH 5.8)
-for 4.0 hr at 27 to 280. Labelled substrates were added with a 100 MAl pipette after adjusting each to 4.5 mc/mmole.
Ethanol extracts include the sum of cpm in hot 80 % and 50 % fractions. The residue is the KOH insoluble and the
KOH soluble material precipitated at pH 1 (including DNA).
Added cpm
1,490,000
1,530,000
1,470,000
1,510,000
Absorption
116,600
65,270
81,380
84,330
C14 recovery (cpm/flask)
Ethanol
extracts
CO2
114,950
320
50,000
3,980
27,400
48,800
14,200
60,800
HC104
Cytosine
Erotic acid
Uracil
Uridine
DL-carbamyl
28,700
2,780
aspartate
34,390
1,490,000
55,380
DL-aspartate
23,100
80,940
1,427,000
* C/U is the specific activity of RNA cytidylic/uridylic acid.
Nevertheless, these results are quite consistent
with those expected if plants synthesize pyrimidine
nucleotides by the pathway proposed in the Introduction. The observed greater conversion of orotic acid
than either aspartic or carbamyl aspartic acids to
-RNA is predicted by its position in the metabolic
sequence, although comparisons are somewhat difficult because only the DL-forms of the latter 2 were
available for studies. The reactions discussed also
indicate that uridylic acid should be labelled prior
to cytidylic acid upon addition of any previous precursor. Table I shows that in all cases where their
labelling was measurable, the specific activity of
uridylic acid was higher than that of cytidylic acid.
In other unpublished experiments with cocklebur, we
found that this C/U ratio increased with time after
addition of orotic acid. In several instances the C/U
-ratios were as high as 1.1 to 1.2 after 16 hours of exposure. To determine whether a similar situation
occurs in bean leaves, a kinetic study with orotic-2-C14
acid was made.
Figure 1 shows the time course for labelling of
both RNA pyrimidine nucleotides over an 11-hour
period. The increase in specific activity of uridylic
acid appears to be linear, but the rate of cytidylic acid
labelling became greater with time. As a result the
C/U ratio increased from 0.43 after 3 hours to 1.0
after 11 hours. The data suggest that if the experiment had progressed longer, C/U ratios significantly
greater than 1.0 would have resulted. Ratios as high
as 2.0 were observed in rat mammary glands, where
the transport of a high specific activity cytidine synthesized in another organ to the gland was suggested
as a possible explanation for the unexpected labelling
(38). Certain reasons to explain ratios greater than
1.0 could also be postulated for our plant tissues, but
the exact mechanism is not known.
Metabolic Products of RNA Precursors. If the
proposed pathway indeed occurs in plants it should be
possible to isolate some of the intermediate products.
Two-dimensional paper chromatograms of the aqueous
ethanol and neutralized perchloric acid extracts were
prepared with this goal in mind. In various ex-
soluble
870
2,410
1,100
1,620
Residue
90
380
275
500
440
420
70
820
RNA
380
8,500
3,800
7,210
2,400
1,220
C/U*
0.60
0.63
0.66
0.63
9
11
0
10
7
a TIME4 (NH)10
4
INcIJUTIO
FIG. 1. Incorporation of orotic acid-2-C14 into RNA
cytidylic acid (C) and uridylic acid (U) in bean leaf
discs, and increase in the C/U ratio as a function of time.
Initially, 2.0 ,uc C14 were added to 230 mg tissue in a
final volume of 5.1 ml 0.01 M pH 5.8 potassium phosphate
buffer.
O
I
a
3
I
periments both bean and cocklebur leaf tissues were
studied, resulting in very similar patterns for metabolites of uracil, cytosine, and orotic acid. As yet,
cocklebur studies with other precursors have not been
made. Figure 2 shows drawings of the autoradiograms made after chromatography of extracts from
bean tissues fed DL-carbamyl-C14-aspartic acid and
2-C'4-labelled uracil, uridine, and orotic acid. The
spots are numbered to correspond to the identifications in table II. No metabolic products of cytosine2-C14 were observed in either cocklebur or bean, after
film exposures comparable to those with the other
metabolites. Only a very dense spot on the films
caused by unchanged cytosine was present. This result substantiates the very poor metabolism of cytosine
shown in table I. It apparently eliminates any chance
that conversion of uracil or other metabolites labelling
RNA cytidylic acid proceeded through cytosine as
an intermediate. Thus, amination must occur at the
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69
ROSS-PYRI M IDI NE NUCLEOTIDE SYNTHESIS
A. CARSAMYL-C" ASPARTATE
B.
URACIL-2-C"4
0
0
To
21
Id
C)
s
0
|
UK-I
I
35
UK-K
0
D. OROTIC ACID-2-C'1
C. URIDINE-2-C'
10r.
7
tn
*6
6 UN-4
30
U5-4
09
QUz~K-s
t.5>
t
09 ,
0
0
0.25
BAW-_
0.50
Rt
0.75
0
0.25
UK-a
0.50
I0
0.75
FIG. 2. Chromatography of metabolites formed in
bean leaf discs from RNA precursors. BAW was run
first in the long direction of the paper. Compounds were
detected by autoradiography and are numbered to correspond to those of table II.
nucleoside or nucleotide level. It is interesting that
the presence of an amino group on cytosine instead
of a keto oxygen as on uracil would lead to such
striking differences in utilization by these plants.
Aspartic acid was converted primarily into other
amino or Krebs cycle acids. Although the data are
not shown, the distribution pattern was rather similar to that reported by Naylor et al. for wheat and
barley leaves (24). Malic acid contained about half
of the total radioactivity detectable on the chromatograms. Glutamic and citric acids were other important metabolites. Another ninhydrin positive
compound was not identified, but was probably threonine. The important observation from these chromatographic studies of aspartic acid metabolites is that
no nucleotides were detected. It is likely that they
were formed but in such low quantities as to escape
detection. Competing reactions involving the added
aspartic acid were apparently much more extensive.
Nucleotides were formed when carbamyl aspartic
acid was added to the leaf discs. Spot 1 contained
principally UDPG, although small amounts of UTP
were also present, as evidenced by chromatography
in other solvents and by paper electrophoresis at pH
3.5 and 5.2. By these methods it was found that any
C14 present in CTP was so low as to escape detection.
The area described by spot 2 was sometimes streaked
between spots 1 and 3, perhaps due to partial hydrolysis in the acidic BAW solvent used in the first dimension. This area contained principally UDP with perhaps very small amounts of CDP present. It is
possible that at least part of these compounds were
formed by destruction of the corresponding triphosphates. Spot 3 contained UMP. Although CMP
was not separated from UMP in these solvents, the
2 were easily resolved by use of IFW in 1 dimension.
No CMP has ever been detected in the UMP area.
Spot 4 contained unmetabolized carbamyl aspartate.
The spot labelled UK-1 immediately above carbamyl
aspartate is probably dihydroorotic acid, but since this
compound was also present in the added solution of
labelled carbamyl aspartate, or was formed from it
during chromatography, it is not possible to say
whether it is a true metabolite of the leaves. Reifer
Table II. Per Cent Distribution of Radioactivity in Compounds Formed from Pyrimidine Nucleotide Precursors
in Bean Leaf Discs
Total radioactivity present in extracts is given in table I. After autoradiograms were made the spots thus detected
were cut out, placed in planchets, and C14 measured.
Precursor
Compound
1. UDPG + UTP
2. UDP
3. UMP
4. Carbamyl aspartate
Orotic acid
51.2
15.1
14.7
...
Uracil
34.5
7.6
14.2
Uridine
34.1
3.0
14.9
...
OL-carbamyl
aspartate
17.9
3.0
9.5
68.0
...
1.0
5.9
0.4.
0.6
2.9
0.6
7.4
30.1
45.4
...
0.5
1.2
...
1.3
6.2
UK-1
..
.
..
]6
0.1
UK-2*
42
0.2
...
UK-3
0.1
2.4
UK-4*
...
G2
* We have not established that UK-2 is the same compound on chromatograms of both orotic acid and uracil metabolites. UK-4 may similarly not be identical in orotic acid and uridine experiments. Only the chromatographic behavior is known to be similar.
5. O-Ureidopropionate
6. Sucrose
7. Uracil
8. Dihydrouracil
9. Uridine
10. Orotic acid
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70
PLANT PHYSIOLOGY
et al. (30) detected labelled UMP, CMP, uracil, uri-
dine, and orotic acid after feeding carbamyl-C14aspartate to excised wheat leaves. We might also
have found the latter 3 compounds had larger quantities of C14 been added, but their amounts relative to
the others would certainly be very low.
Leaf discs incubated with uracil, uridine, and orotic acid formed the same labelled nucleotides as those
fed carbamyl aspartate (spots 1 to 3 of fig 2 A-D).
The relative C14 content of each labelled area is given
in table II. Since partial hydrolysis of the di- and
triphosphates and of UDPG occurred during elution
and/or subsequent concentration and rechromatography, it was not possible to report the exact radioactivity in UTP and UDPG, each of which was present in spot 1. These data are, however, consistent
with results of others (38,17) who have found C14
in these nucleotides after adding 1 or more of the same
labelled substrates. The low content of C14 in cytidine compounds relative to uridine nucleotides has
been observed in various animal tissues (38,17).
Bieleski and Laties (6) reported very small amounts
of CTP and CDP present in potato tubers and were
unable to detect CMP. Buchowicz and Reifer (9)
found only about 10 % as much radioactivity in CMP
as in UMP after adding labelled orotic acid to wheat
leaves.
Uracil was metabolized into several other compounds, including ,8-ureidopropionic acid, sucrose,
and 2 others not identified. Unknown number 2
(UK-2) moved almost identically to carbamyl aspartate in the BAW, t-BKW solvents, but uracil-2-C14
would not be expected to label carbamyl aspartate.
It has not been found on enough chromatograms to
allow further studies as to its nature. UK-3 was
ninhydrin positive and ran very near serine, yet it is
not this amino acid. It was also found in the cocklebur extracts reported previously (32). Ureidopropionic acid is an intermediate in uracil catabolism
in various organisms (35), and has been identified in
cocklebur leaf discs (32), Longleaf pine tissue cultures (3), and rape seeds (15) following uracil feeding. The detection of labelled sucrose was surprising,
and its mechanism of synthesis from uracil is not
understood. Sucrose can be formed from urea (12).
We have looked carefully for evidence of uridine
formation from uracil, since uridine was an intermediate in UMP formation from uracil in the Ehrlich ascites tumor (35). Such uridine has not been
detected on our chromatograms of bean or cocklebur
extracts. This suggests, but certainly doesn't prove,
that uridine does not participate in synthesis of UMP
from uracil. This problem will be considered further
below.
On the other hand, uridine was converted to uracil
in relatively large quantities (table II), perhaps by
a uridine phosphorylase. Orotic acid labelled both
uracil and, to a greater extent, uridine. Conversion
of erotic acid to uracil and uridine was reported in
pine embryos by Barnes and Naylor (3) and in wheat
leaves by Buchowicz and Reifer (7). The latter authors' evidence indicated that either
or both were
intermediates in formation of UMP from orotic acid.
They thus proposed that formation of orotidine-5'phosphate could be at least partially bypassed in wheat
blades. They were unable to detect either this compound or orotidine in the extracts. Such a bypass
is considered unlikely for RNA synthesis in bean and
cocklebur leaves for 4 principal reasons. Instead, the
labelling of uridine and uracil by orotic acid probably followed, not preceded, synthesis of UMP. First,
we have found previously (32) that RNA synthesis in
cocklebur leaves from orotic acid was blocked by addition of 6-azauracil. Azauracil is known to be converted metabolically to azauridine-5'-phosphate, which
then inhibits orotidine-5'-phosphate (OMP) decarboxylation (27, 33). This implicated OMP in RNA
formation in this plant. Carbon dioxide release from
orotic acid was inhibited to about the same degree
as was its incorporation into RNA, further showing
that the principal sensitive reaction in RNA synthesis
is at OMP decarboxylase (32).
Secondly, decarboxylation of orotic acid appears
to be dependent on PRPP. Experiment 2 of table
III shows nearly a 6-fold increase in CO2 release
from orotic acid-carboxyl-C14 in bean leaf homogenates when 10-4 M PRPP was added, and a 13-fold
stimulation by 10-3 M PRPP. Formation of uracil or
uridine directly from orotic acid as proposed by
Buchowicz and Reifer (7) would likely not require
PRPP, whereas if OMP is an intermediate, PRPP
is essential (22).
Thirdly, table IV shows that when azauracil or
azauridine was added to inhibit OMP decarboxylation, accumulations of OMP and orotidine, as well
as of orotic acid, were noted in bean and cocklebur
leaf discs fed orotic acid-carboxyl-CI4. Without inhibitor only orotic acid and traces of orotidine (in
beans) were found. (We previously observed only
orotic acid accumulation when these inhibitors were
added to cocklebur leaves (33), but less sensitive
detection techniques were used). The majority of
the C14 not converted to CO2 was accounted for in
Table III. Stimulation of C1402 Release from Orotic
Acid-Carboxyl-C'4 in Bean Leaf Homogenates
by PRPP
Eighteen g buds and young leaflets from 6-week-old
bean plants were frozen, then homogenized 3 min in 45 ml
cold 0.05 M Tris, pH 7.4, using a Servall Omni mixer at
top speed. Homogenate was filtered through cheesecloth,
and 4.0 ml then added to flasks with other additions to
make 0.001 M MgCl,2 PRPP as indicated, and 1.0 Ac
orotic acid (5.3 mc/mmole). Final volume was 5.55 ml.
Flasks were incubated in darkness, with shaking, 3.8 hr
at 270. C1402 collected in 3 %o KOH and measured by
direct plating. All values are means of 3 flasks.
Molarity PRPP
None added
0.0001
0.001
C1402 recovery (cpm/flask)
Expt. 1
Expt.2
150
101
450
573
...
1300
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ROSS-PYRI MIDINE
Table IV. Formiation of Orotidine and Orotidine
A1onophosphate in Bean, and Cocklebutr Leaves
front Orotic Acid-Carboxyl-C'4
To 290 mg fr wt of cocklebur leaf discs, 0.6 ,uc orotic
acid-carboxyl-C14 was added. Tissue was incubated in
10 ml of 0.01 M potassium phosphate, pH 5.8, for 3 hr,
then killed in hot 80 %o ethanol. Ethanol extracts were
cochromatographed with the known compounds and autoradiographed.
Bean leaf discs were incubated in 10 ml 0.05 M Tris,
pH 7.4, containing 2.0 ac orotic acid. All values are
means of duplicate flasks. Specific activity of orotic
acid was 5.3 mc/mmole.
C14 in compounds (cpm/flask)
0.003 M
0.003 Ai
No inhibitor 6-azauracil 6-azauridin2
Cocklebur
Orotic acid
Orotidine
Orotidine-5'phosphate
Bean
Orotic acid
Orotidine
Orotidine-5'phosphate
1,140
Not found
2,660
235
7,910
170
Not found
Not found
730
...
6,150
360
...
32,900
3,970
Not found
...
625
orotic acid, perhaps because the equilibrium catalyzed
by OMP pyrophosphorylase strongly favors orotic
acid (22). The orotidine might have arisen from
phosphatase action on OMP.
Finally, the possibility of prior conversion of orotic
acid to uracil before incorporation into RNA is eliminated by the nearly 2-fold greater ability of orotic
acid than uracil to label the RNA. Uridine involvement cannot be disproved as convincingly by this latter argument alone, since it was almost as efficiently
used for RNA synthesis as was orotic acid.
Orotic acid was converted into 16-ureidopropionic
acid (carbamyl 18-alanine) in both cocklebur andl
bean leaf discs (table II). This compound is normally considered to be a catabolic product of uracil.
The corresponding product of orotic acid catabolism
is carbamyl aspartic acid (,8-ureidosuccinic acid),
not identified in our orotic acid experiments, although it was apparently produced in pine embryos
and tissue cultures from orotic acid (3). UK-2
could be carbamyl aspartate, based on its chromatographic movement, yet it appears to be the same
compound as was found in the uracil experiments.
UK-4, consistently found in orotic acid experiments
and often in the uridine studies, had chromatographic
properties close to those of uridine. It was not ninhydrin positive.
The formation of 18-ureidopropionate suggests
that catabolism of orotate may have occurred after
its conversion to UMP and uracil. Evidence that
this is an important pathway in cocklebur leaf tissues,
at least, is that C1402 release from orotic acid-2-C1t
is inhibited by 6-azauracil, known to lead to an inhibition of OMP decarboxylase (27, 32), thus blocking UMP formation. Similar reactions were proposed for orotate breakdown in rat livers (37).
N
71
UCLEOTIDE SYNTHESIS
Influence of Uridine on Uracil Incorporation into
RNA. If uracil is initially converted to uridine prior
to labelling uridine nucleotides, simultaneous addition
of uridine with uracil-2-C14 should dilute the incorporation of C14 into RNA. Table V shows results
of an experiment with bean leaf discs in which a concentration of uridine equal to that of added uracil-2C14 (5 X 10 6 Ai) decreased the specific activity of
the RNA by 22 % while a 10-fold higher uridine concentration reduced it by 82 %. This effect was not
due simply to an inhibition of uracil absorption by
the uridine, although some decrease in total C14 recovered in the tissues was observed. The results
suggest uridine was an intermediate in conversion of
uracil into RNA and appear, therefore, contrary to
the chromatographic data where no uridine was detected after addition of uracil.
To clarify this problem, 5 X 10-4 AI uridine was
added with labelled uracil to form a trapping pool
for any uridine produced metabolically. Portions of
the alcohol soluble tissue extracts were chromatographed 1-dimensionally in the neutral -ethanolammonium acetate solvent and 2-dimensionally in
BAW and t-BKW. Autoradiography of the latter
chromatograms did show radioactivity in the uridine.
Its specific activity was 1,400 cpm/,umole. It represented only 0.57 % of the total C14 present on the
chromatograms. It is assumed that this uridine arose
directly from uracil although it is also possible that it
was formed from dephosphorylation of UMP.
Figure 3 shows the distribution of radioactivity
in compounds along the 1-dimensional chromatograms. Where no uridine was added with the labelled
26
-
UMP+UDPG
24
22
20
14
~~~~~~~~~~~~~~~URACIL
0
.12
0
6 10
4~~~~~~~~~~~~~~~~~~~'u
A~~~~~~~U
DISTANCE FROM ORIGIN (cm)
FIG. 3. Distribution of radioactivity in metabolites of
uracil-2-C14 in the presence of 5 X 10-4 M uridine (dotted
line) or in its absence (solid line). Chromatography
was performed with the neutral ethanol-ammonium acetate solvent on paper strips. Strips were cut into 1 cm
sections, eluted, and counted in the planchet counter.
Values are means of 4 samples each.
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72
PLANT PHYSIOLOGY
uracil, most of the C14 was in nucleotides (3 peaks
closest to the origin) and little in residual uracil
(last peak). The addition of uridine decreased incorporation of uracil into the nucleotides and caused
its accumulation in the tissues. This result shows
that the inhibition of incorporation of uracil into RNA
by uridine (table V) did not occur simply because
Table V. Inhibition of RNA Synthesis from
Uracil-2-Cl4 by Uridine in Bean Leaf Discs
Approximately 640,000 cpm uracil was added at a final
concentration of 5 X 1-6 M to 10 ml 0.01 M potassium
phosphate, pH 5.8. Ten leaf discs were incubated 4 hr
in near darkness. Each value is a mean of 4 flasks.
Uridine
added
None
5 X 10-6 M
5 X 10-5 M
cpm
absorbed
57,400
52,000
36,500
cpm in
CO2
21,800
22,200
15,100
RNA C14
(cpm/mg)
10,200
7,940
1,840
uridine increased the total nucleotide pool, thus diluting the radioactivity in nucleotides derived from uracil. Instead, the effect is on conversion of uracil into
the nucleotides. This result, combined with the above
observation that uracil labelled uridine, is suggestive
that uridine is a true intermediate in RNA synthesis
from uracil. The necessary enzyme, uridine phosphorylase, has apparently not yet been demonstrated
in plants. These reactions of uracil and uridine do
not appear to be important in the normal synthetic
pathway for RNA, which apparently involves only
orotic acid as a free pyrimidine.
Conclusions
Both the experiments comparing the rate of incorporation of pyrimidine nucleotide precursors into
RNA and the chromatographic studies suggest that
much of the pathway proposed for other organisms
is probably valid for higher plants as well. Specifically, RNA synthesis from aspartic, carbamyl
aspartic, and orotic acids was consistent with their
place in the proposed reaction sequence. The latter
2 were converted to the required uridine nucleotides,
UMP, UDP, and UTP, in easily detectable quantities,
although kinetic data for the order of their labelling
was not obtained. In addition, UDPG was labelled
by these 2 precursors. Cytidine nucleotides were
probably formed but, if so, the quantities were very
small compared to the uridine nucleotides.
When orotic acid-carboxyl-C'4 was provided, the
leaf discs formed small quantities of OMP and orotidine. The addition of 6-azauridine aided these findings because of a resulting inhibition of OMP decarboxylase, thus allowing some accumulation of
these compounds. Even though OMP is an essential intermediate in RNA synthesis, it too is present
only in small amounts, probably because of a rapid
decarboxylation to UMP. This likely explains why
Buchowicz and^ Reifer (7) were unable to find it in
wheat leaves. As far as we are aware, this is the
first demonstration of either OMP or orotidine in
higher plants. The large stimulation of C140, loss
from carboxyl-labelled orotic acid by PRPP is
strongly suggestive of an OMP pyrophosphorylase
enzyme synthesizing OMP from orotate, as was found
by Lieberman et al. (22) in yeast. In the crude
homogenate we used, however, other explanations for
the result of PRPP addition are not ruled out.
These leaf discs also converted uracil and uridine
into RNA. Uracil catabolism was very rapid and
this is undoubtedly one reason it was less efficiently
used in RNA synthesis than was uridine. The data
suggest uridine is an intermediate in uracil anabolism,
as in the mammals studied (14). The failure of cytosine to significantly label the RNA was also consistent with animal results (4). In fact, cytosine was
hardly metabolized by either bean or cocklebur
leaves.
Formation of 18-ureidopropionic acid from orotic
acid as well as from uracil and uridine suggests the
possibility that orotate catabolism normally occurs
after conversion to the uridine nucleotide level. No
detectable 8-ureidosuccinic acid (carbamyl-aspartic
acid) was formed when orotate was fed. Previous
evidence to support this interpretation was obtained
in cocklebur leaf discs (32). It thus appears that
orotate breakdown is not simply a reversal of its synthesis.
Summary
Leaf discs taken from beans, Phaseolus vulgaris
var. Idaho 111, or cockleburs, Xanthiun pennsylvanicum Wall., converted several compounds into RNA
uridylic and/or cytidylic acids. These, listed in the
approximate order of efficiency as RNA precursors
in the bean, were: orotic acid, uridine, carbamyl
aspartic acid, uracil, and aspartic acid. Traces of
C14 from cytosine also appeared in RNA but the extent was only about one-tenth that of uracil incorporation. All except aspartic acid and cytosine labelled
soluble uridine nucleotides, including a compound with
properties of uridine diphosphate glucose. Cytosine
was very poorly metabolized. Formation of orotidine-5'-phosphate and orotidine from carboxyl-labelled
orotic acid was demonstrated, especially where azauracil or azauridine was added to cause their accumulation. This result, combined with a large stimulation
of C140, loss from orotic acid-carboxyl-C14 by 5phosphoribosylpyrophosphate, indicates a normal
function for orotidine-5'-phosphate in plants.
It was concluded that the results support much
of the proposed pathway of pyrimidine nucleotide
synthesis described for other organisms.
Some of the results indicate orotic acid is principally catabolized after its conversion to uridine
nucleotides, then by the normal pathway for uracil
breakdown through ,8-ureidopropionic acid.
Acknowledgments
The technical assistance of Janet Thomas, Joseph
Miller, and John Wolcott is appreciated. Robert Starr
kindly performed the tissue combustion analyses.
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ROSS-PYRIMIDINE NUCLEOTIDE SYNTHESIS
Literature Cited
1. ANONYMOUS. 1956. Ultraviolet absorption spectra
of 5'-ribonucleotides. Pabst Laboratories Circular
OR-10. Pabst Brewing Company, Milwaukee.
2. BANDURSKI, R. AND S. MAHESHWARI. 1962. Nucleotide incorporation into nucleic acid by tobacco
leaf homogenates. Plant Physiol. 37: 556-60.
3. BARNES, R. AND A. NAYLOR. 1962. Formation of
13-alanine by pine tissues supplied with intermediates
in uracil and erotic acid metabolism. Plant
Physiol. 37: 171-75.
4. BENDICH, A., H. GETLER, AND G. BROWN. 1949. A
synthesis of isotopic cytosine and a study of its
metabolism in the rat. J. Biol. Chem. 177: 565-70.
5. BIELESKI, R. AND R. YOUNG. 1963. Extraction and
separation of phosphate esters from plant tissues.
Anal. Biochem. 6: 54-68.
6. BIELESKI, R. AND G. LATIES. 1963. Turnover rates
of phosphate esters in fresh and aged slices of potato tuber tissue. Plant Physiol. 38: 586-94.
7. BUCHOWICZ, J. AND I. REIFER. 1961. The conversion of erotic acid to pyrimidine derivatives in
plant material. Acta Biochim. Polon. 8: 25-34.
8. BUCHOWICZ, J. 1963. Incorporation of [2-14C] uracil into polynucleotides in homogenates of wheat
seedlings. Acta Biochim. Polon. 10: 301-07.
9. BUCHOWICZ, J. AND I. REIFER. 1962. The synthesis
of pyrimidine nucleotide derivatives in plant material using [6-14C] erotic acid. Acta Biochim.
Polon. 9: 63-70.
10. CANELLAKIS, E. 1957. Pyrimidine metabolism.
III. The interaction of the catabolic and anabolic
pathways of uracil metabolism. J. Biol. Chem.
227: 701-09.
11. CANELLAKIS, E. 1957. Pyrimidine metabolism. II.
Enzymatic pathways of uracil anabolism. J. Biol.
Chem. 227: 329-38.
12. CLoR, M., A. CRAFTS, AND S. YAMAGUCHI. 1963.
Effects of high humidity on translocation of foliarapplied labelled compounds in plants. II. Translocation from starved leaves. Plant Physiol. 38:
501-07.
13. CRAWFORD, A., A. KORNBERG, AND E. SIMMS. 1957.
Conversion of uracil and orotate to uridine-5'-phosphate by enzymes in Lactobacilli. J. Biol. Chem.
226: 1093-1101.
14. CROSBIF, G. 1960. Biosynthesis of pyrimidine nucleotides. In: The Nucleic Acids, Vol III. p
323-48. E. Chargaff and J. Davidson, eds. Academic Press. New York and London.
15. EVANS, W. AND B. AXELROD. 1961. Pyrimidine
metabolism in germinating seedlings. Plant
Physiol. 36: 9-13.
16. FINK, K., R. CLINE, R. HENDERSON, AND R. FINK.
1956. Metabolism of thymine (methyl-C'4 or
-2-C14) by rat liver in vitro. J. Biol. Chem. 221:
425-33.
17. HURLBERT, R. AND V. POTTER. 1954. Nucleotide
metabolism. I. The conversion of erotic acid-6C14 to uridine nucleotides. J. Biol. Chem. 209:
1-21.
18. KAMMEN, H. AND R. HURLBERT. 1958. Amination
of uridine nucleotides to cytidine nucleotides by
mammalian enzymes: Role of glutamine and guanosine nucleotides. Biochim. Biophys. Acta 30:
195-96.
19. KAPOOR, M. AND E. WAYGOOD. 1961. Enzymes of
erotic acid metabolism in wheat embryos. Plant
Physiol. suppl. 36: v.
73
20. KING, J. AND D. WANG. 1961. Biosynthesis of pyrimidines and their derivatives in the wheat plant.
Plant Physiol. suppl. 36: v.
21. LIEBERMAN, I. 1956. Enzymatic amination of uridine triphosphate to cytidine triphosphate. J. Biol.
Chem. 222: 765-74.
22. LIEBERMAN, I., A. KORNBERG, AND E. SimMS. 1955.
Enzymatic synthesis of pyrimidine nucleotides orotidine-5'-phosphate and uridine-5'-phosphate. J. Biol.
Chem. 215: 403-15.
23. LIEBERMAN, I., A. KORNBERG, AND E. SIMMS. 1955.
Enzymatic synthesis of nucleoside diphosphates and
triphosphates. J. Biol. Chem. 215: 429-39.
24. NAYLOR, A., R. RABSON, AND N. E. TOLBERT. 1958.
Aspartic-C'4 acid metabolism in leaves, roots, and
stems. Physiol. Plantarum 11: 53747.
25. NEUMANN, J. AND MARY E. JONES. 1962. Aspartic
transcarbamylase from lettuce seedlings: Case of
end product inhibition. Nature 195: 709-10.
26. PALADINI, A. AND L. LELOIR. 1952. Studies on
uridine diphosphate glucose. Biochem. J. 51:
426-30.
27. PASTERNAK, C. AND R. HANDSCHUMACHER. 1959.
The biochemical activity of 6-azauridine: interference with pyrimidine metabolism in transplantable
mouse tumors. J. Biol. Chem. 234: 2992-97.
28. REICHARD, P. 1959. The enzymatic synthesis of
pyrimidines. Advan. Enzymol. 21: 263-94.
29. REIFER, I., J. BUCHOWICZ, AND K. TOEZKO. 1960.
The synthesis of the pyrimidine ring from L-carbamyl aspartic acid in excised blades of wheat seedlings. Acta Biochim. Polon. 7: 29-38.
30. REIFER, I., J. BUCIIOWICZ, AND K. TOEZKO. 1961.
Metabolism of L-carbamyl aspartic-C14 to pyrimidine derivatives in excised wheat leaves. Acta
Biochim. Polon. 8: 377.
31. Ross, C. 1962. Nucleotide composition of RNA
from vegetative and flowering cocklebur shoot tips.
Biochim. Biophys. Acta 55: 387-88.
32. Ross, C. 1964. Influence of 6-azauracil on pyrimidine metabolism of cocklebLr leaf discs. Biochim. Biophys. Acta 87: 564-73.
33. Ross, C. 1964. Metabolism of 6-azauracil and its
incorporation into RNA in the cocklebur. Phytochem. 3: 603-07.
34. SALZMAN, N., H. EAGLE, AND E. SEBRING. 1958.
The utilization of glutamine, glutamic acid, and
ammonia for the biosynthesis of nucleic acid bases
in mammalian cell cultures. J. Biol. Chem. 230:
1001-12.
35. SCHULMAN, M. 1961. Purines and pyrimidines.
In: Metabolic Pathways, Vol. II. p 389-457. D.
Greenberg, ed. Academic Press. New York and
London.
36. STARR, R. AND C. Ross. 1964. A method for the
determination of carbon in plant tissues. Anal.
Biochem. 9: 243-46.
37. VON EULER, L., R. RUBIN, AND R. HANDSCHUMACHER.
1963. Fatty livers induced by orotic acid. II.
Changes in nucleotide metabolism. J. Biol. Chem.
238: 2464-69.
38. WANG, D. AND A. GREENBAUM. 1962. The incorporation of [2-14C] glycine and [6-14C] orotic acid
into the acid soluble ribonucleotides and ribonucleic
acids of the rat mammary gland. Biochem. J. 83:
626-32.
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