Download Photosynthesis in the Higher Plant, Vicia.faba

Plant Physiol. (1974) 53, 491-495
Photosynthesis in the Higher Plant,
Vicia.faba
III. SERINE, A PRECURSOR OF THE TRICARBOXYLIC ACID CYCLE'
Received for publication August 9, 1973 and in revised form November 26, 1973
S. SHERRILL KENT,2 FREDERICK D. PINKERTON, AND GARY A. STROBEL
Department of Plant Pathology, Montana State University, Bozeman, Montana 59715
ABSTRACT
Evidence is presented to support the hypothesis that serine,
rather than 3-phosphoglycerate of the Calvin cycle, is a
precursor of the tricarboxylic acid cycle during photosynthesis
by the higher plant, Vicia faba. Identification of the serine intermediate is based upon a unique C1 > C2 > C3 isotope distribution for that metabolite following the fixation of "CO2.
This labeling pattern, while incompatible with an origin either
in the Calvin cycle or the glycolate pathway, satisfies a critical
criterion for the 3-carbon precursor of the anomalously labeled
organic acids. The predominant carboxyl carbon atom labeling
of serine reflects either a mixing of two pools of that metabolite, ie., C1 = C2> C3 and C1 > C2 = Cs, or a higher order of
complexity in its synthesis. An anomalous C1 = C2 > C3 < C
distribution for aspartate, however, suggests an origin by the
carboxylation of a 3-carbon intermediate related to serine
which has a C1 = C2 > C3 distribution. The latter distribution
has been proposed for the serine intermediate of the postulated
formate pathway. This pathway is described by the generalized
metabolic sequence: C02 -+ formate - serine -* organic acids.
Corresponding carbon atom distributions for citrate (C1 > C2),
aspartate (C2 > C3), and serine (C > Cs) belie a precursorproduct relationship with alanine (C2 = C3), which is a molecular parameter of the Calvin cycle product, 3-phosphoglycerate.
The present investigation describes a precursor-product relationship between photosynthetically generated serine and
the anomalously labeled (15) organic acids of the higher plant,
Vicia faba. A previous communication (16) supported the hypothesis that formate arises de novo and is the direct precursor
(via serine) of the methylene carbon atoms of the organic acids,
particularly the acetate methylene carbon atom of citrate. The
supposition that 3-carbon intermediates of the Calvin cycle are
precursors of the organic acids was precluded on the basis of
kinetic, inhibitor, and intramolecular isotope distribution studies. Indirect evidence for de novo formate synthesis was given
by the high levels of 14CO2 fixed into formate under conditions
which inhibited the Calvin cycle, the selective inhibition by
arsenite of the Calvin cycle but not of formate synthesis, and
the preferential fixation of respiratory 'CO3 into formate, as
opposed to fixation into the Calvin cycle. A reversible reaction
involving the direct reduction of CO2 to formate was compatible with a specific, mutual competition between CO2 and formate for assimilation, and with high ratios of 'H/"C in the
organic acid methylene carbon atoms resulting from 'H/"Cformate assimilation.
Indirect evidence for the existence of a unique serine precursor of the organic acids was based upon precursor-product
relationships, inhibitor studies, and theoretical considerations
(16). First, exogenously supplied 'H-formate labeled serine as
a primary product and the methylene carbon atoms of the
organic acids as final products. Calvin cycle-related products,
particularly glycine and alanine, were not labeled with tritium.
Second, arsenite induced an accumulation of photosynthetic
"4CO2 label in formate and concomitantly blocked the labeling
of the methyl (but not the carboxyl) carbon atom of the acetate
fragment of citrate, i.e., citrate (C-1 ,2). The inhibitor did not
alter the equal labeling of carbon atoms 2 and 3 of the Calvin
cycle product, alanine, thus precluding a precursor-product
relationship with the acetate fragment of citrate. Third, formate carbon, via carbon atom 3 of serine, is at least a potential
precursor of the methylene carbon atoms of the organic acids.
A key aspect of the previous study (16) concerned the distribution of label in seine after photosynthesis in 14CO2. To
establish this metabolite as the unique 3-carbon precursor of
the anomalously labeled organic acids, an isotope distribution
(exempting masking by uniformly labeled serine of the glycolate pathway or predominant carboxyl carbon atom labeling
from phosphoglyceric acid-derived serine) was predicted to be
one in which C, = C2 > C3 (16). Evidence for this unique distribution is presented.
MATERIALS AND METHODS
General Procedures. Vicia faba (var. English Windsor)
plants were grown in sandy soil supplemented with Hoagland's
microelements. The water supply contained ammonium nitrates
and phosphates. The greenhouse temperature was 20 C
5
C. Plant leaves were used for experimentation after 4 to 6
weeks of growth.
Photosynthetic "2CO fixation conditions, preparation and
fractionation of leaf extracts, Chl determinations, colorimetric
assays, and the isolation of citrate and aspartate have been
described (15, 16). Citrate, alanine, and aspartate were degraded by the method of Kent (14). Radioactivity was determined with a Model 3320 Packard Tri-Carb liquid scintillation
counter; absolute count rate was calculated by the channels
for quench correction (2).
1This investigation was supported by the National Science ratio
Degradation of Serine. A new method for the degradation
Foundation Grant GB 23918 and United States Department of
of stereoisomers of serine was developed by Kent for the presAgriculture Cooperative States Research Service Grant 216-15-23.
ent study and is described elsewhere (17). This method elimi2 To whom reprint requests should be addressed.
491
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492
problems of cross
contamination between carbon atoms
poor yields for carbon atom 3 of serine
(19) which are encountered in the periodate procedure of
Sakami (21). In brief detail, isotopically and molecularly pure
L-serine is enzymically converted to pyruvate by a rat liver
preparation of L-serine dehydratase (EC 4.2.1.13). The pyruvate is oxidatively decarboxylated with H20, to acetate and
CO2; and, in a series of coupled reactions with acetate kinase
(EC 2.7.2. 1), phosphotransacetylase (EC 2.3.1 .8), and citrate synthase (EC 4.1.3.7), the 2-carbon fragment is condensed with nonradioactive oxaloacetate to form 1,2-14C-citnates
2 and
Plant Physiol. Vol.
KENT, PINKERTON, AND STROBEL
3,
as
well
as
rate.
In a single test tube reaction, isotopically pure citrate is
quantitatively decarboxylated to 3 moles of CO2 and 1 mole
of pentabromoacetone. The "4C-pentabromoacetone is selectively extracted into heptane; and its specific radioactivity,
which is equivalent to the specific radioactivity of serine (C-3),
is determined directly in the citrate colorimetric assay. While
the specific radioactivities of the remaining two carbon atoms
of serine may also be determined directly, such extensive manipulations have not proved necessary. The isotope distribution in serine may be calculated with an error of less than 3%
from the specific radioactivities of the original 14C-serine,
1, 2-"4C-citrate, and "4C-pentabromoacetone.
Isolation of Serine and Alanine. Radioactive leaf extracts
were successively fractionated by anion (5) and cation (12) exchange chromatography. The neutral amino acid fraction was
hydrolyzed (6 N HCl, 100 C, 10 hr) and passed through a
1.2 X 3 cm resin bed of Dowex 1 (formate) overlayed with 2
of Norit A to remove aromatic amino acids and hydrolyzed glutamine and asparagine. Serine and alanine, eluted
in 10 ml of water, were twice resolved by paper chromatography in 0.1% NH3-phenol (v/v) (6). Phenolics were removed
by adsorption on Norit A. Isotopic purity was established by
the appearance of single peaks of radioactivity coincident with
those of appropriate standards on a Packard Tri-Carb stripscanner. Molecular purity with respect to ninhydrin reactivity
was confirmed by the appearance of single peaks on a Beckman
amino acid analyzer before and after addition of carrier amino
acid.
cm
RESULTS AND DISCUSSION
Kinetics of Photosynthesis in 14CO2, In the previous study
of V. faba (16), the time course of photosynthesis was examined under conditions which favored the production of formate. The synthesis of this 1-carbon compound was generally
observed in plants which had been grown during the summer
months. Under optimal conditions, the rate of fixation of
"CO2 into formate was one-third of the total fixation rate
which was 7.5 ,moles/hr ,umole of Chl at a light intensity of
0.02 langley/min. When photosynthesis was inhibited 80%
by arsenite, as much as 65% of the "CO2 label appeared in
formate. Plants which were grown during the winter months
failed to accumulate formate. This cyclic phenomenon was
observed over a 3-year period. All known variables, except for
temperature and light, were eliminated. Although a complete
description of the seasonal formation of formate is beyond the
scope of the present study, temperature is suspected to be the
more critical variable. Formate was assayed by methods already described (15).
In the present investigation, the kinetics of photosynthesis
in 14CO were examined under conditions which do not favor
formate synthesis. Although summer plants were used, the
temperature at which the plants were grown is comparable to
that of the winter plants described previously (16). Otherwise,
all experimental conditions, particularly low light intensity
53, 1974
and saturating CO2 tensions, were identical to the original time
course experiment described by Kent (16).
Excised V. faba leaves were infiltrated with distilled, deionized water and illuminated in an atmosphere of 1 % 'C02-air
for successive periods of 4, 8, 15, 22, and 35 min. The light
intensity at the leaf surface was 0.02 langley/min. The temperature of the photochamber was 22 C. To minimize any
metabolic variations, four leaves were used for each fixation
period.
As shown in Figure 1, the rate at which `CO2 label is fixed
into both the neutral compounds, i.e., the major Calvin cycle
products, and the organic acids are constant. Formate was not
detected. In the previous study (16) when formate was produced during photosynthetic "CO2 fixation, "plateau-rise"
curves were observed for both formate and the organic acids;
the rate of fixation of "CO2 into the products of the Calvin
cycle was constant.
Intramolecular Isotope Distributions. The supposition that
a uniquely labeled serine molecule is the precursor of the tricarboxylic acid cycle in V. faba was substantiated by analyzing
the intramolecular isotope distributions of four key intermediates: citrate, aspartate, alanine, and serine. The first two compounds are metabolites of or related to the tricarboxylic acid
cycle. Anomalous labeling of citrate during photosynthesis has
been reported previously by Kent (15). Aspartate was selected
as a molecular parameter of malate. The latter compound was
found to be a major end product of exogenously assimilated
3H formate (16). Alanine essentially represents a control. In
V. faba (16), and in other higher plants (1, 8, 9, 20), this metabolite has been shown to possess an intramolecular isotope
distribution identical to that of 3-P-glyceric acid, the primary
product of the Calvin cycle. The anomalous labeling pattern
for citrate is characterized by a C, > C2 isotope distribution in
the acetate moiety, which is classically derived from the oxidative decarboxylation product of pyruvate, i.e., carbon atoms
2 and 3. The unequal labeling of the acetate fragment of citrate
is shown in Table I. The isotope ratio of citrate (C-1/C-2) was
1.8 from 4 to 35 min of photosynthesis. Since the presumed
precursor of the cited acetate moiety, namely 3-P-glycerate,
has an isotope distribution in which C, > C2 = C3, a C, = C2
distribution would ordinarily be predicted. While 3-P-glycerate
was not analyzed directly in the present study, its typical isotope distribution is, nevertheless, reflected by the isotope distribution in alanine (Table II), which is metabolically removed
from the presumed pyruvate precursor of the tricarboxylic acid
cycle by a single transamination step. The absence of a meta-
4 8
15
22
35 4 8
TIME (MIN)
15
22
35
FIG. 1. Comparative kinetics of "CO2 fixation into the Calvin
cycle products, i.e., the neutral compounds, and the organic acids.
The organic acids are comprised of malate, citrate, glutamate,
and aspartate. Other acids of the tricarboxylic acid cycle possess
less than 1% of the total "CO2 fixed. Formate was not detected
as a photosynthetic intermediate in this experiment.
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SERINE METABOLISM
Plant Physiol. Vol. 53, 1974
Table I. Distributiont of Isotope in Citrate after
Photosynzthetic 14C02 Fixation
The unequal labeling of citrate carbon atoms I and 2 is anomalous. While pyruvate is the classical precursor of citrate C-1,2, an
equal labeling of carbon atoms 2 and 3 of alanine (Table II) precludes a precursor-product relationship.
Distribution
Carbon Atom
C-1
C-2
C-3,4
C-5,6
Citrate (dpm/
umole
4 min
8 min
15 min
22 miin
42
21
12
25
1,340
43
22
14
21
36
24
17
23
41
24
15
20
41
20
18
21
2,500
5,850
6,320
10,300
35 min
Table II. Distribulioni of Isotope in Alaizine after
Photosynithetic 1 4CO? Fixationi
The average specific radioactivity ratio of alanine (C-2/C-3) is
0.99. This value conforms to the Cl > C2 = C3 distribution of a
3-carbon compound derived from 3-P-glycerate of the Calvin
cycle. For the degradation of the U/'4C-alanine control, the percentage of distribution of isotope was 34.1, 32.8, and 33.1 for
carbon atoms 1, 2, and 3, respectively.
Distribution
C-1
C-2
C-3
Alanine (dpm/
,umole)
8 min
rate which is classically derived from pyruvate. In the actual
experimental isolation of "4C-serine, the mixing of pools of
Calvin cycle serine (C1 > C2 = C3) and glycolate-pathway serine
(C1 = C2 = C3) with the proposed pool of formate-pathway
serine (C1 = C2 > C3) would tend only to reduce the degree
but not the critical fact of unequal labeling for carbon atoms
2 and 3 (Scheme I). In fact, for the present investigation, the
mechanism whereby C-3 of serine attains a lower specific radioactivity than C-2 is immaterial. A simple exchange of carbon at
the C-3 position of serine with a pool of N5,N10-formyl tetrahydrofolic acid of lower specific radioactivity than serine (C-2)
is not precluded by the data. Evidence for a mechanism involving a direct reduction of CO2 to formate with the incorporation of formate carbon into serine (C-3) has been described
by Kent (16). If, however, the exchange mechanism is correct
and de novo formate synthesis is invalid, then this study raises
the question as to whether glycolate-pathway serine or Calvin
cycle serine is the key precursor of the organic acids.
Given the precision and accuracy of the "4C-degradation
methods, the difference in the isotope ratios between citrate
(C-1 /C-2) and serine (C-2/C-3) is significant. The fact that the
isotope ratio for citrate (1.8) is greater than that for serine
(1.5) may be accounted for by the mixing of formate-pathway
serine with Calvin cycle serine or glycolate-pathway serine in
the extraction procedure, or both. The identification of the
various metabolic pools of serine and the possibility of a C1 >
C2 > C3 isotope distribution is the subject of a future investigation. It is presently stressed that a possible admixture of serine
Table III. Distributionz of Isotope in Serine after
Carbon Atom
4 min
493
15 min
22 min
64
18
18
63
18
19
62
19
19
60
20
20
51,000
104,000
489,000
215,000
bolic relationship between 3-P-glycerate-derived alanine and
citrate (C-1 ,2) was more emphatically described by the observation that arsenite specifically blocked the incorporation of
14CO2 isotope into citrate (C-2) without altering the equal labeling of alanine (C-2,3) (16).
The single photosynthetic intermediate which satisfies the
criterion of the 3-carbon precursor of the organic acids in V.
faba is serine (Table III). A C1 > C2 > C3 isotope distribution
is not predicted for a serine metabolite derived either from
3-P-glycerate in the Calvin cycle or from glycine in the glycolate pathway. In the first case, the serine which may be produced from the Calvin cycle is predominantly labeled in the
carboxyl carbon atom position. Carbon atoms 2 and 3 are
always equally labeled. A C1 > C2 = C, labeling pattern for
serine in algae, at least, has been interpreted to involve a
synthesis from 3-P-glyceric acid (3, 11, 23). In the second case,
the pool of serine which arises in the glycolate pathway from
the condensation of 2 moles of uniformly labeled glycine is itself uniformly labeled (10, 20). Furthermore, the ultimate fate
of glycolate-pathway serine involves a conversion to sugars.
Significantly, both of the cited metabolic pools of serine are
equally labeled in carbon atoms 2 and 3. In the present study
of V. faba, the serine which is isolated after photosynthesis in
"'CO, is not only unequally labeled in carbon atoms 2 and 3,
but the isotope distribution exactly fits that distribution anticipated for the 3-carbon precursor of the acetate moiety of cit-
Photosy,ithetic '4CO2 Fixation
The magnitude of the unequal labeling of carbon atoms 2 and
3 of serine satisfies the critical criterion of a 3-carbon precursor
of the acetate fragment of citrate, i.e., citrate (C-1,2). This criterion is in accord with the observation that 3H formate labels
serine as a primary product and the methylene carbon atoms of
the organic acids, particularly citrate (C-2), as final products (16).
The percentage of distribution of isotope in the U/'4C-serine
control was 32.0, 33.5, and 34.5 for carbon atoms 1, 2, and 3, respectively. The control ratio of 0.97 compares favorably with a
theoretical ratio of 1.00.
Distribution
Carbon Atom
4 min
C-i
C-2
C-3
Serine (dpm/,Amole)
63
22
15
5,800
8 min
15 min
22 min
62
23
15
13,000
55
26
19
24,400
55
26
18
36,600
C-Isotope Distributions for Serine
0 C0
0
02
0 C3
GIyco/ote
Pathway
*000wC
00
eeoc1
*
C2
0 C3
Formote
Pothwoy
.
00
C2
003
0 C3
Calvin
Mixture
Cycle
Scheme I.
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C2
c
*
494
KENT, PINKERTON, AND STROBEL
Plant Physiol. Vol. 53, 1974
Aspartate was also labeled with tritium at the methylene carbon
atom position, and is presumably related to malate through a
common oxaloacetate intermediate. This relationship is, at
least, supported by comparable isotope ratios, i.e., C-1 ,4/C-2,3
for aspartate (1.34) and the oxaloacetate fragment of citrate
(1.44). A summary of the unusual metabolism which has
emerged in these studies of V. faba is shown in Scheme II.
The preceding information describes a system of interrelated
Distribution
anomalies involving citrate, aspartate, malate, and serine. These
Carbon Atom
anomalies are not accounted for by Calvin cycle carbon me35 min
22 min
8 min
15 min
tabolism. An involvement of part of the tricarboxylic acid cycle
in this system was demonstrated by the identical intramolecular
isotope distributions for both citrate and glutamate. Further29
28
27
27
C-1
more, a citrate to glutamate direction of metabolism was indi24
25
27
26
C-2
cated (15). Presently, the involvement of the remaining portion
17
16
19
17
C-3
of the tricarboxylic acid cycle in the postulated formate path31
29
28
30
C-4
way system is in question.
44,500
26,400
5,600
18,000
Aspartate (dpm/
A final point which must be considered is the relationship of
1ymole
the uniquely labeled serine observed in V. faba to the serine
which arises in the glycolate pathway during photorespiration.
The
glycolate pathway involves a sequence of reactions from
pools in this study does not alter the general conclusions. If glycolate
to glyoxylate to glycine to serine, which is then cona
as
it is the case that the serine (C-2/C-3) isotope ratio is lower
verted
to
sugars via 3-P-glycerate (22). This sequence and the
result of pool mixing, then it is probable that only one meta- intramolecular
localization of enzymes which catalyze the reacbolic pool of serine, possessing a C, = C2 > C, distribution, is
the major precursor of the organic acids. This supposition is tions have been well documented in Tolbert's laboratory (22).
fact that
directly supported by the analysis of the isotope distribution Of particular relevance to the present study is theconditions
photorespiration is a consequence of environmental
in asparatate (Table IV). This intermediate possesses a "C dis- involving
high light intensities, low CO, tensions, and high
tribution in which C, = C2 > C, < C4. For reasons cited below, oxygen tensions.
These conditions are essential for the movethis distribution must be considered anomolous. However, the ment
the glycolate pathway (26). Studies of
of
carbon
through
distribution for the first three carbon atoms is particularly relevant in that a C, = C2 > C, distribution was predicted for serine the labeling patterns of key intermediates have shown that in
in the previous study of V. faba (16). The distribution at least higher plants glycolate (10, 11, 25) and glycine (10, 11) are
supports the contention above that formate-pathway serine is
FORMATE PATHWAY INTERMEDIATES
the major, if not the only precursor, of the tricarboxylic acid
for
in
V.
the
distribution
aspartate, particcycle
faba. Otherwise,
ularly the unequal labeling of carbon atoms 2 and 3, is anomaC,OOH
C, OOH
lous. If aspartate were to arise from the carboxylation of a
3-carbon compound related to 3-P-glyceric acid (C, > C2 =
N -Ca
N -Ca
C.), the cited carbon atoms would be equally labeled. In fact,
C13HOH
C;10- w
the isotope distribution for aspartate is compatible with the
I0
to
related
the
3-carbon
uniquely
compound
carboxylation of a
C400H
(53
labeled serine product described above. Furthermore, if this
serine metabolite is the exclusive precursor of aspartate, the
Aspartate, Malate
Serine
aspartate (C-2/C-3) isotope ratio should be the same as that
[Cl= Ca >C.]Theor
>=CCe C4]Obs
[CICC
for citrate (C-i/C-2). That the ratio for aspartate (1.5) is, in
fact, less than that for citrate (1.8) may be accounted for by
[Ca
>CG]Obs
randomization via a symmetrical intermediate. Alternatively,
the Calvin cycle may contribute in a secondary fashion to the
4-carbon acids.
)
(C02)
©) COOH
In addition, an alternative mechanism which could account
for the isotope distribution in aspartate would be mediated by HOOCa-CR- Ca-OH
HOOCa-GC- a
malate synthetase. This glyoxylate cycle enzyme would conCCR8 -NN
Is
0
dense unequally labeled acetate with equally labeled glyoxylate
COOH
COOH
to produce the ultimately. observed isotope distribution in
aspartate. Malate synthetase, however, was not detected in
leaves of V. faba (Pinkerton, F. D. and S. S. Kent, unpublished
Glutamate
Citrate
observation) and apparently is not present in the leaves of
[Ca :>CS] Obs
[Ca > Ce]Theor
several other higher plants (24). The presence of isocitritase
would not account for the isotope distribution in serine. The
Scheme II. The a- and p-carbon atom assignments correspond
predominant carboxyl carbon atom labeling of the glyoxylate to the original a- and ,8-carbon atoms of serine. The 8-carbon atom
product would produce serine with a C, > C2 = Ca distribution of serine originates from "C-formate. The tritium labeling, desigif that glyoxylate were to enter into glycolate-pathway me- nated by ), results from the light-dependent assimilation of 'Htabolism, which is unlikely.
formate (15). Relative carbon atom specific radioactivities which
In the previous study, Kent (16) found that serine and result from photosynthetic "CO2 fixation are shown in brackets.
malate were the primary and major end products, respectively, The theoretical "C-isotope distribution in glutamate has been
of a light-dependent 'H-formate assimilation by V. faba leaves. reported by Burns et al. (4) for tobacco.
Table IV. Distribution of Isotope in Aspartate after
Photosynthetic '4 C02 Fixation
A Cl = C2 > C3 < C4 isotope distribution for aspartate is
anomalous for reasons discussed in the text. The unequal labeling
of carbon atoms 2 and 3 implicate serine (Table III) rather than
alanine (Table II) as the metabolically related precursor of aspartate.
CI
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SERINE METABOLISM
Plant Physiol. Vol. 53, 1974
equally labeled at all times of photosynthesis in "4CO2. Serine,
which arises by the condensation of 2 moles of glycine in the
mitochondrial transhydroxymethylase complex (7, 18), is also
uniformly labeled (10, 20). In algae, the carboxyl carbon atom
of serine is predominantly labeled after short periods of photosynthesis. This phenomenon has been attributed to a secondary
synthesis from 3-P-glycerate (3). Of particular relevance to the
present study is the fact that the a- and /-carbon atoms of
the serine intermediate, which arises in the glycolate pathway,
are always equally labeled. There is no precedence for unequal
labeling except in V. faba. In the latter case, photorespiration
and glycolate pathway activity have been repressed by carrying
out photosynthesis at low light intensities and high CO2 tensions
(13). Quite reasonably, the C1 > C2 > C. isotope distribution
for the serine metabolite which is isolated from V. faba may be
detected only under nonphotorespiratory conditions.
A major distinction between the glycolate pathway and the
recently postulated formate pathway is that the former produces equally labeled serine which is a precursor of the sugars
in the Calvin cycle while the latter produces unequally labeled
serine which is the precursor of the organic acids in the tricarboxylic acid cycle.
Acknowledgment-The authors wish to express their appreciation to K.
Hapner for performing the amino acid analyses.
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