ACCUMULA'HON 9F CITRIC AGED EN FRUIT

Thesis for the Degree of Ph D.
MEI-SEAN STAT-E UNiVERSITY
MOSHE TISHEL
1957

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thesis entitled

ACCUMULATION 0F CITRIC ACID IN FRUIT

presented by
Moshe Tishel

has been accepted towards fulfillment

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ABSTRACT
ACCUMULATION OF CITRIC
ACID IN FRUIT

by Moshe Tishel

The acetone powders of the orange, lemon, tangerine,
grapefruit, strawberry, and tomato fruits (all citric acid
accumulators), the apple and sweet lime fruits (malic acid
accumulators), the orange flavedo (an oxalate accumulator)
and the Bartlett pear (a fruit with high malic and citric
acid content) were assayed for the activity of enzymes asso-
ciated with citrate metabolism.

Aconitase, isocitric dehydrogenase, and malic dehy-
drogenase showed activities which did not differ considerably
from one fruit to another. Citric synthase varied markedly
from fruit to fruit, but no correlation between citric acid
content and this activity was apparent. The activities of
isocitric dehydrogenase (DPN dependent) and isocitritase were
not detected in these tissues.

Acetate 2-14C was injected to detached green tomatoes
and apples. Half an hour after injection the incorporated

C was associated with citric acid in both fruits.

Moshe Tishel

After twenty—four hours citric acid remained the
most labeled acid in the tomato while malic acid was most
highly labeled in the apple,

Glucose-U.L. l--4C, was injected into detached green
tomatoes and apples. In the apple, citric acid became
highly labeled said one hour after injection but malic acid
became the dominantly labeled acid after twenty—four hours.
In the tomato an unknown compound incorporated a major por-
tion of the 14C from glucose within thirty minutes after
injection and declined with time with a concurrent increase
in labeled citric and malic acids and after twenty—four
hours most of the label was in the citric acid fraction
and none in the unknown.

A double label experiment with 14C and 32P indicated
that the unknown acid must be a phosphorylated compound.
Homogenate studies established the dependence of this un—
known on ATP for its biosynthesis. The label of glucose
U.L.—14C as well as glucose 1—14C and glucose 6-14C was
found in this compound, eliminating decarboxylation as a
step in its biosynthesis. In addition acetate 2—14C did

not serve as a precursor.

Acetate 2—14C was fed to apples and tomatoes, the

Moshe Tishel
distribution of radioactivity between particles precipitated
at 22,000 x g and the supernatant fraction was compared. In
the apple, 33% of the organic acid radioactivity was found
in the particles compared with only 5% in the tomato. Citric
acid was the only labeled acid in the apple particles.

The organic acid profile in the fruit tissues examined
does not correlate with the enzyme activity profile. Labeled
precursor experiments corroborate the enzyme survey finding.
Finally the intracellular distribution of newly synthesized
organic acids indicates the posfibility of compartmentation

as a factor in acid accumulation in fruits.

ACCUMULATION OF CITRIC

ACID IN FRUIT

by

Moshe Tishel

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science

1967

c3Z0’5?

67/???“ 7

07

ACKNOWLEDGEMENT

The author is greatly appreciative of the encourage—
ment, guidance and constructive criticism given to him by
Dr. P. Markakis.

The author wishes to thank Dr. D. R. Dilley for his
constructive help and criticism. The assistance given to
him by Dr. C. L. Bedford, Dr. J. R. Dugan and Dr. L. R.
Brunner is appreciated.

The author would like to thank Dr. R. C. Nicholas

for his advice in the preperation of this manuscript.

ii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENT . . . . . . . . . . . . . .

TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . iii

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . V
LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi
I. INTRODUCTION . . . . . . . . . . . . . . . . . 1
II. LITERATURE REVIEW . . . . . . . . . . . . . . . 3
Acid Accumulation . . . . . . . . . . . . . . 3

Acid Translocation . . . . . . . . . . . . . 4

002 Fixation . . a . . . . . . . . . . . . . 5

The Tricarboxylic Acid (TCA) Cycle . . . . . 9
Compartmentation . . . . . . . . . . . . . . 10

III. MATERIAL AND METHODS . . . . . . . . . . . . . 14
Preparation of Enzymes for Assay .
Assay of Enzymes . . . . . . . . . . .

Aconitase . . . . . . . . . . . . . . . . . l4
Isocitric dehydrogenase (TPN dependent) . . 15
Isocitric dehydrogenase (DPN dependent) . . 15
Malic dehydrogenase . . . . . . . . . . . . 15
Citric synthase . . . . . . . . . . . . . . 16
Isoctritase . . . . . . . . . . . . . . . . 16
Controls . . . . . . . . . . . . . . . . . l6

Acid Extraction and Determination . . . . . . 17
Administration of Radioactive Material . . . 17
Determination of Radioactive Material . . . . 19
Paper Chromatography . . . . . . . . . . . . 20
Isolation of Sub-cellular Particles . . . . . 20

iii

Page
IV. RESULTS AND DISCUSSION . . . . . . . . .

Theories . . . . . . . . . . . . . . . . . . . 22
Enzymic Imbalance . . . . . . . . . . . . . . 23

Comparison of Enzyme Activity in Various
Fruits . . . . . . . . . . . . . . . . . . 24

Aconitase . . . . . . . . . . . . . . . 25
Isocitric Dehydrogenase (TPN Dependent). 25
Isocitric Dehydrogenase (DPN Dependent). 28
Malic Dehydrogenase . . . . . . . . . . 28
Citric Synthase . . . . . . . . . . . . 31
Dialysis . . . . . . . . . . . . . . . . 34
Isocitritase . . . . . . . . . . . . . . 34

Radioactive IsotopeExperiments . . . . . . . 38

Incorporation of Acetate 2—14C . . . . . 39

Incorporation of Uniformly Labeled
Glucose —14C . . . . . . . . . . . . . 41
Homogenate . . . . . . . . . . . . . . . 48

Attempts to Identify the Unknown .
Double Label Experiments . . . . . . . . 50
Column Chromatography . . . . . . . . . 50
Paper Chromatography . . . . . . . . . . 50

Compartmentation . . . . . . . . . . . . . . . 52

V. SUMMARY AND CONCLUSIONS . . .

LITERATURE CITED . . . . . . . . . . . . . . . . . . 59

iv

LIST OF FIGURES

Figure Page
1. The Injection of Fruit Under Vacuum . . . . . . l9
2. Aconitase Activity in Some Fruits . . . . . . . 26

3. Isocitric Dehydrogenase (TPN Dependent) Activity
of Some Fruits . . . . . . . . . . . . . . . 29

4. Malic Dehydrogenase Activity of Some Fruits . . 30

5. Citric Synthase Activity of Some Fruits . . . . 32
6. Effect of Dialysis on Citric Synthase Activity

of Apples and Oranges . . . . . . . . . . . . 35
7. Incorporation of the Label of Glucose— UL -14C

Into the Organic Acids of Detached Tomato

Fruits, 1 Hour After Injection . . . . . . . 44

8. Incorporation of the Labels of Glucose- UL -
14C and phosphate —32P Into the Organic Acid!
of Detached Tomato Fruits; 2.5 Hours After
Injection . . . . . . . . . . . . . . . . . . 46

LIST OF TABLES
Table Page
I Aconitase Specific Activity . . . . . . . . . . 27

II Isocitric Dehydrogenase (TPN Dependent)
Specific Activity . . . . . . . . . . . . . 27

III Citric Synthase Specific Activity .
l4 14

IV Incorporation of C of Acetate 2- C Into the
Organic Acids of Detached Green Tomato Fruits 40

. . . . . . 33

V Incorporation of 14C of Acetate 2-14C Into the
Organic Acids of Detached Preclimacteric
Northern Spy Apple Fruits . . . . . . . . . . 40

. 4
VI Incorporation of 1 C of Glucose-U.L. £4C Into
Organic Acid of Detached Apple Fruits . . . . 43
VII Incorporation of the Label of Glucose U.L. l4C
Into Organic Acids in Detached Green Tomato
Fruits . . . . . . . . . . . . . . . . . . . 45

VIII Incorporation of 14C From Glucose-14C Into the
Organic Acids by Tomato Homogenate . . . . . 49
. l4 14
IX Incorporation of C of Glucose U.C. - C and
PO —32P Into Organic Acid of Detached Green
Tomato Fruits . . . . . . . . . . . . . . . . 51

X Rf Values of Some Phosphorylated Compounds . . 51
. l4 14
XI Incorporation of C From Acetate -2— C Into

the Organic Acids of Particulate and
Supernatant Fracfions of Apple and Tomato Fruits 55

vi

I. INTRODUCTION

Organic acids are important constituents of food.

They contribute to the flavor of foods and act as food pre—
servatives. On the other hand, acids take part in undesirable
changes in food processing. Chlorophyll degradation, off—
flavors, and browning may be caused by acids during processing
and storage.

Citric acid is an important food additive. It is
produced commercially either by microbial fermentation or
obtained from fruits such as lemon fruits which contains
about six percent citric acid. The biochemical and physi-
ological process by which citric acid accumulates in plants
is therefore interesting both from the academic and practical
standpoints.

Plant tissues, in contrast to animal tissues, tend
to accumulate one or more organic acids. Citric acid is the
major acid igujuice of citrus fienét, tomato, and strawberry
fruits. Malic acid is the main acid of apples. cherries,
and quinces.

The accumulation of a certain acid is not a property

1

2
characteristic of a family or a genus or even of a given
species. Anjou pear accumulates malic acid, whereas the
Barlefl:variety contains about equal amounts of both citric
and malic acids. Even in a given fruit such as the naval
orange, the flavedo accumulates oxalate while citric is the
main acid in the juice. There are now indications that dif—
ferent acids may be distributed unequally within the same
cell.

This study was conducted to elucidate the process of
citric acid accumulation in fruits. A dual approach was em—
ployed: a) a comparative study of the enzymatic activity of
tissues of high and low citric acid content, and b) a com-
parative study of the incorporation and metabolism of organic

acid precursors in the two types of tissues.

II. LITERATURE REVIEW

Acid Accumulation

 

Malic and citric acids are most frequently the major
components of the organic acid fraction in fleshy fruits.
Citric predominates in orange, lemon, strawberry, gooseberry,
and tomato; whereas malic is accumulated in apples, plums,
and cherries (Ranson, 1965). Other acids are also found in
significant amounts in plant tissues. Roberts and Martin
(1954) reported that aconitic acid is the major acid compon-
ent in sugar cane. It is the most plentiful acid in young
shoots of wheat and rye as well as in maize roots and
<xfleoptile (Ranson, 1965). MacLennan and Beevers (1964) have
shown that about 95% of the aconitate which accumulates in
maize roots supplied with acetate was of the trans-configur—
ation type.

Grapes accumulate tartaric acid in addition to malic
(Peynaud and Maurie, 1953) and Phaseolus coccineus accumu—
lates malonic acid as well as malic and citric acid5(Bently,
1952). The fruits of blackberry, and the leaves of EEXET

phyllum and Kalanchoe accumulate isocitric acid (Pucher gt.

 

3

4
al., 1949; Whiting, 1958). A recent review of the distri-
bution of organic acids in higher plants has been published
by the U.S.D.A. (Buch, 1960).

An interesting aspect of acid metabolism is the
Crassulacean acid metabolism which has been reviewed by
Ranson and Thomas (1960). In the dark, malate is produced
in the cytoplasm and accumulates in the vacuole. In the
light, the vacuole is depleted of malic acid. Citrate gen—
erally varies in phase with malate, but with markedly smaller
amplitude. Isocitric acid, often the acid present in high-
est concentration, shows very little diurnal change (Vickery,
1952).

Although the types and the quantities of organic
acids are supposedly determined genetically, Kenworthy and
Harris (1964) noted that the geographic location determines
the acid profile of apples. This observation is supported
by Rasmussen and Smith (1960) who noted that oxalate level
in orange leaves responds to changes in Ca and K while

levels of citric acid remain unchanged.

Acid Translocation

 

It is often stated that the acids which accumulate

in fruits are largely synthesized in the leaves and trans-

5
located into the developing organs. Pucher §t_al, (1937,

1949) observed that during germination of Lupinus angusti—

 

folius the acid content decreased in the cotyledons and
increased in the seedling axis. This tendency was shown
for each main acid component, malic, citric, phosphoric and
an unknown.

Allsop, according to Ranson (1965), found that oxalic,
malic, and citric acids decreased in the rhizomes of sprout—
ing rhubarb and increased in the shoots developing from them.
Each of these instances supplied indirect evidence that acids
were translocated unchanged from one organ to another.
Kursanov (1963), however, found that malic and citric were
not translocated from assimilating cells. He pointed out
that in the phloem sap sugars were the main soluble compon—
ent, although aliphatic acids, amino acids and other com-
pounds were found. Webb and Barley (1964) stated that
stachyose and sucrose were the compounds which were mostly

translocated in various plants.

992 Fixation

Wood and Werkman (1938) reported that dark fixation

of CO2 occurs in bacteria. Bonner and his co—workers (1948)

6
postulated and later confirmed CO2 fixation into malate
which accumulated in the leaves of Crassulaceae. This was
confirmed by Wood (1952), Gregory 22 31, (1954), and Moyse
(1955).

Allentoff, Phillips and Johnston (1954) reported
that most of the CO2 which was fixed by apple fruit was in—
corporated into malic acid. Ninety-nine percent of the
label was incorporated in the carboxyl groups.

Bean and Todd (1960) reported on light and dark

CO fixation in the young orange fruit. The flavedo exhibited

2
high photosynthetic activity in contrast to the juice ves—
icles. In the dark, however, the juice vesicles were far
more active in dark fixation than any other tissue. Malic
and citric acids were almost equally labeled and they were
the major labeled compounds.

CO2 fixation in citrus fruits was confirmed by Bogin
and Wallace (1966) and Bogin (1966) . Phosphoenolpyruvate (PEP)
carboxylase, malic enzyme and isocitric dehydrogenase were
considered as the enzymes which were responsible for this
financing
Clark gt 31. (1961), while examining CO fixation

2

in orange and avocado, showed that either phosphoenolpyruvate

7

or ribulose-S—phosphate can serve as the CO acceptors. They

2
demonstrated also the presence of a polyphenolic inhibitor
of CO2 fixation in avocado leaves.
Several enzymes were postulated as responsible for
dark CO2 fixation:
1. Malic enzyme (OChoa et_al,, 1947).

+ TPNH === Malate + TPN. This

Pyruvate + C02 2

enzyme is widely distributed in plant tissues
(Vennesland and Conn, 1952).

2. PEP carboxylase catalyses the following reaction:
PEP + C02====Oxalacetate + Pl‘ This enzyme was
first described by Bandurski and Greiner (1953)
and is very widely distributed in plant tissue
(Mazelis and Vennesland, 1957). According to
the latter authors malic dehydrogenase which
catalyses the reduction of oxalacetate to malate
is also widespread in plants. Together these
two enzymes may account for the fixation of CO2
into malate.

3. Mazelis and Vennesland (1957) also reported that

another enzyme, PEP carboxykinase, was widely

distributed in plants.

8

PEP + CO2 + ADP ==soxalacetate + ATP.

4. Isocitric dehydrogenase is found in many plant
tissues (Vennesland and Conn, 1959).

d- Ketoglutarate + CO2 + TPNH2 =2 isocitric + TPN

Moyse and Jolchime (1956) suggested that isocitrate
which is accumulated may be synthesized by carbox—
ylation of ci-ketoglutarate.

Walker (1957) argued that the equilibrium of malic
enzyme is unfavorable toward malic acid synthesis while syn-
thesis of oxalacetate by PEP carboxylase is virtually irre—
versible. Walker and Brown (1957) pointed out that PEP car—

boxylase has a high affinity to CO It will reach optimum

2.

activity at 0.5% CO While carboxylation of pyruvate by the

2
malic enzyme reaches a maximum at 30%.C02.
Buhler gt_al, (1956)evaluated the physiological

importance of CO2 fixation in tomato. They injected 2—14C

pyruvate into tomato and determined the radioactivity in
each of the carbons in malic and citric acids. They concluded

that although CO fixation on pyruvate does exist it is of

2
little physioloqical importance. This supports Walker's data

but does not shed light on the role of CO2 fixation via PEP

or ci—ketoglutarate.

9

Clark and Wallace (1963) attempted to correlate CO2

fixation to the amount of total acidity in citrus fruits.

They found that sweet lime had the highest CO incorporation,

2

while lemon had the lowest, and orange was intermediate.

The relative acid content was found to be 1:11:37 for sweet
lime, orange and lemon,respectively, which is in the reverse
order to the CO2 fixation capacity. The authors concluded

that CO2 fixation may not be the controlling mechanism for

acid accumulation.

The Tricarboxylic Acid (TCA) Cycle

 

 

There is little doubt today that the TCA cycle is
present in plant cells. There is ample evidence to justify
the view that the TCA cycle is the major route of pyruvate
utilization in plant, as in animal cells. (Beevers, 1961).
Evidence has accumulated that the TCA cycle may play a role
in acid accumulation.

Markakis and Embs (1964) demonstrated that labeled
sugars may be converted to radioactive organic acids in de-
tached strawberry fruits.

Deshpande and Ramakrishnan (1961) suggested that

the TCA cycle enzymes may be involved in regulating acid

10

accumulation in fruit. In their work on Garcinia fruit they
demonstrated that citric acid production was concurrent with
a ten-fold increase in citric synthase activity and a simul—
taneous decrease in aconitase activity. On ripening, when
the amount of citric acid decreased, citric synthase activ—
ity diminished and citric desmolase appeared.

Jangaard, et_al,, demonstrated that ATP caused a
20-fold increase in the apparent Km of citric synthase to-
ward acetyl CoA; therefore, ATP may act as a regulator of

acid accumulation.

Compartmentation

 

Plant cells have a large vacuole which frequently
accounts for 90% of their volume. Stiller (1959) calcu—

lated that if malic acid in Bryophyllum leaves were confined

 

to the cytoplasm, its concentration would be 7—8 M; but if
it were in the vacuole also, its average concentration would
be 0.25 M, a more credible value. Her experimental results
also indicate compartmentation of malate, since the malate
formed from l4CO2 remained asymmetrically labeled, but

asymmetrically labeled malate provided to the tissue from

outside became randomized. The vacuole need not be consid—

11
ered as the only storage reservoir of metabolites, however,
since a degree of compartmentation within the cytoplasm it—
self has been invoked as a rational explanation for results
of labeling patterns (Porter and May, 1955).

McLennan gt 21, (1963) designed experiments to
determine the existence and extent of compartmentation.
They argued that if a labeled compound isolated from a bio—
logical tissue had a higher specific activity than its
labeled precursor, then only a fraction of the precursor
pool is involved in the interconversion. Using a 1—14C
acetate, they demonstrated that when the specific activity
of the carbon dioxide respired by masize root tips became
constant, indicating that equilibrium had been reached in
the turnover pools of the acids of the Krebs cycle, there
were great differences in the specific activities of the
individual acids. The differences were ascribed to the
existence of pools of acids not in ready equilibrium with
turnover pools, but which mixed with them during extraction.
Evidence from maize root shows that, as vacuolation occurs,
the relative amount of acid not in turnover pools increased

markedly.

Some workers examined the ability of isolated mito-

12
chondria to incorporate metabolites. They found that the
mitochondria were impermeable to externally added citrate
(Schneider, Striebich and Hogeboom, 1956; Bartley and Davies,
1953; Amoore, 1958). It was also demonstrated that various
chemicals can influence the accumulation and oxidation of
citrate by isolated mitochondria (Chappel, 1964; Williams,
1965; Gamble, 1965; Meyer and Tager, 1966). L-malate in
the absence of inorganic phosphate increases the permeabil—
ity of mitochondria towards citrate. When inorganic phos—
phate is added the nitochondria become impermeable to citrate.
Dinitrophenol and ADP + Pi block the entrance of citrate
into the mitochondria. Max and Purvis (1965) found that
succinate enhances the entry and exit of citrate into mito—
chondria.

Temperatures close to 00 will retard metabolite
incorporation by mitochondria and decrease metabolic activ—
ities of mitochondria. Accumulation of citrate by mito—
chondria is energy linked (Max and Purvis, 1965) which
explains some of the effects of various compounds on citrate
accumulation.

Cerijo-Santalo (1966) examined the conditions which

cause mitochondrial swelling and found that isotonic solu—

13
tion and neutral pH favor retention of mitochondrial struc—
ture. Swelling is promoted by hypotonic solutions with

subsequent leakage of substances.

III. MATERIAL AND METHODS

Preparation g£_Enzymes for Assay

 

 

The enzymes were prepared as acetone powder according
to Nason (1955). This method has the advantage of giving a
chlorophyll—free preparation with a minimum contamination of
gum and resin—like material. At least in one case (aconitase
activity in apple) the enzyme could be demonstrated in the
acetone powder, but not in the apple homogenate, due probably
to a polyphenol inhibitor (Hulme et_gl,, 1964). Extraction
of the enzymes was accomplished by suspending 300 mg powder
in 10 ml phosphate buffer pH 7.1. After 15 minutes the pow—

der was filtered and the clear filtrate was assayed.

Assay 9£_Enzymes

All enzyme assays were performed at room temperature.
The specific reaction condition are given in legend to appro-
priate figures.

Aconitase

 

This enzyme catalyses the following reaction:

citric acid .-.=: isocitric acid
. . .W .
Cis-aconitic aCld

14

15
Aconitase was assayed by the method of Anfinsen
(1955). This method is based on the conversion of citrate
and isocitrate to aconitate, and the spectrophotometric
determination of the latter at 240 mu.

Isocitric dehydrogenase (TPN dependent)

 

 

The following reaction is being catalysed by this
enzyme:

Isocitrate + TPN +==dcketoglutarate + CO2 + TPNH2
Kornbergs'method (1955) was used for the assay of TPN depen-
dent isocitric dehydrogenase. The reduction of TPN in the

presence of isocitrate was followed at 340 mu.

Isocitric dehydrogenase (DPN dependent)

 

 

The following reaction is catalysed by this enzyme:

Isocitrate + DPN + ;=2 OK: ketoglutarate + CO2 + TPNH2
Two methods were employed. In both,reduction of DPN in
presence of isocitrate was followed at 340 mu. While Korn—

berg (1955) uses a neutral pH, Davies (1953) uses pH 9.

Malic dehydrogenase

 

Malic + DPN e== oxalacetate + DPNH2
Here the oxidation of DPNH in the presence of oxalacetate is

followed at 340 mu. According to Ochoa (1955) the equilib-

rium lies in the direction of malate formation.

16

Citric synthase

 

This enzyme catalyses the following reaction:
Oxalacetate+acety1 CoA e== citrate + CoA
Ochoa's method (1955) for assaying citric synthase activity
is based on the coupling of citric synthase with malic dehy—
drogenase. DPNH formation in the presence of malate and
acetyl CoA is followed at 340 mu.

Isoctritase

 

Isocitritase catalyses the following reaction:

Isocitrate e== sccinate + glyoxylate

Two methods were employed, both were based on the
formation of glyoxylate from isocitrate. According to Car-
penter and Beevers (1959) the reaction is run at pH 7.6 and
the color of the 2.4 dinitrophenylhydrazones of glyoxylate
is read at 445 mu. According to Olson (1959) the reaction
mixture is buffered to pH 6.0 and the semicarbazones of
glyoxylate are followed at 252 mu.
Controls

All enzyme assays were run with two controls: 1)
the reaction mixture with one reagent missing and 2) the

reaction mixture with a boiled enzyme.

17

Acid Extraction and Determination

 

The fruit acids were extracted with acidified boil-
ing water, and depectinized with ethanol. The chlorophyll
was removed by petroleum ether extraction. The partially
purified mixture of the acids was then passed through an
anion exhanger (Dowex—1X8). The column was washed by ca
60 m1 deionized H20 to remove neutral compounds. Subse-
quently the acids were separated by gradient elution using

acetic and formic acids. The fractions were dried and

titrated with NaOH solution (Markakis §E_al,, 1963).

Administration 2£_Radioactive Material

 

 

Two techniques of administering radioactive pre—
cursors to the fruit were employed.

1. Radioactive compounds were injected by syringe
into single tomatoes or apples. The compounds
were: uniformly labeled glucose -14C, glucose
-l—14C, glucose —6-14C, 2-14C acetate, and 32P-
phosphoric acid. About 1 uC per fruit was
administered. At appropriate time intervals,

which are specified in "Results and Discussions,"

the whole fruit was macerrated in boiling water.

18
The extraction of the acids was executed
as described in the previous section.
For the compartmentation study experiments
the following technique was used:

The needle of a syringe was inserted
into the center of the fruit (tomato and
apple) and the cylinder was fitted through
the rubber stopper of a vacuum dessicator.
The radioactive solution was placed in the
syringe and the dessicator was evacuated
through a second hole in the stopper (Figure l).

The radioactive solution was made from
1 uC of 2-C acetate dissolved in 5 m1 of 0.35
M mannitol.

The solution diffused into the flesh of
the fruit by displacement of the intercell-
ular air during evacuation. Approximately
1 ml of solution per 15 gram of fruit was
administered (Dilley, 1967). At appropriate
time intervals single whole fruitswere
homogenized and fractionated as is described

in the previous section.

19

 

 

 

 

Vacuum
_[ ii:—
Syringe
Fruit
rf#”’/##fr##,,s
r‘] r Dessicator

 

 

 

 

Figure 1. The injection of fruit under vaccuum.

Determination 9£_Radioactive Material

 

 

Aliquotes of the fractionated organic acids were
dried on disposable aluminum planchets as an infinitely thin
layer and counted by a scaler, automatic changer, and time
printing instrument. (Nuclear Chicago Company)

A liquid scintillation spectrometer was employed for
simultaneous counting of 32P and 14C. For this a Packard
tri—carb liquid scintillation spectrometer Model 3003 was
used with a window setting of 50-250 for carbon and 350-00
for phosphorus. The scintillation liquid was prepared as

follows:

160 gm Naphthalene
9 gm BBOT 2,5 [2— (5 tertbutylbenzoxozolyl)
Thiophane]
75 gm Cab-O-Sil

20
770 ml P- dioxane
770 ml Xylene
.462 ml Ethanol
Fifteen m1 of the scintillation liquid was employed with 0.5
ml of aqueous samples.

The Vanguard automatic chromatogram scanner, Model

880 was used for counting paper chromatograms.

Paper Chromatography

 

The compound was run ascendingly along side with the
markers on a Whatman No. I paper. The solvent system was
ethyl— methylketone: water saturated butanol:propanol:formic
acid 45:25: 5:20(v/v) (Gerlach §E_§l,, 1955).

After the solvent front traveled the entire length,
the paper was dried at room temperature and irrigated a
second time with the same solvent. The following spray was

used: 5 m1 of 60%.W/W HClO 10 ml of l N HCl, 25 ml of 4%

4’
W/V (NH4) 2M004, made up with water to 100 ml (Block gE_§l,,

1958).

Isolation p£_Sub-cellular Particles

 

 

All steps in the preparation of mitochondria were
conducted at 0—20C. Since the pH of the ground tomato sus—

pension was about 4, it was necessary to neutralize the acid

21
during grinding. This was achieved by grinding the tomatoes
with 1.5 X their weight 0.1 M phosphate buffer pH 7.5, 0.4 M
sucrose, which brought the slurry to pH 7.0-7.2. Ten grams
of tomatoes were ground and immediately diluted to 50 ml
with 0.1 M phosphate buffer pH 7.0, 0.4 M sucrose. In a
similar manner the apple tissues acids were neutralized by
adding an equal weight of 0.4 M phosphate buffer pH 7.5,
0.25 M sucrose. The suspension was diluted to 50 ml with
0.05 M phosphate buffer pH 7.0, 0.4 M sucrose. The homogen—
ate was filtered through cheese cloth and milk filter paper

and centrifuged at 22,000 x g for 10 minutes.

IV. RESULTS AND DISCUSSION

Theories

It is well known that the Krebs cycle Operates in
all living organisms. While the intermediate acids of the
cycle can be found in catalytic amounts in animal cells,
one or more di— or tri—carboxylic acids may be accumulated
in high concentration in plant cells.

The amount of acids in plants and the acid profile
are probably genetically determined, since they are unique
to any given plant tissue.

Two theories have been advanced to explain this
phenomenon:

l. The enzymic imbalance theory according to
which the acid profile of the fruit is de—
termined by a corresponding enzymic profile.
2. Preferential compartmentation. According

to this theory the characteristic acid pro-

file results from a specific mechanism

involving removal of a certain acid from

its biosynthetic site to a storage site,

22

23
and a consequent shifting of equilibrium
toward further synthesis of this acid.
While the first theory emphasizes the role of the enzymes,
the second stresses the migration of the acids as cause for
acid accumulation. These two possibilities are not mutually

exclusive, however.

Enzymic Imbalance

 

It may be assumed that the enzyme activity of the
tricarboxylic acid cycle (TCA) in animal cells is carefully
balanced and controled and the accumulation of acids is con—
sequently avoided. In plants, however, it is conceivable
that enzyme activity is unbalanced which may lead to high
acid concentrations. Hence citric acid may be accumulated
either as a result of a high activity of citric acid forming
enzymes or because of a low activity of citric acid degrad—
ing enzymes. Citric synthase which catalyses the formation
of citric acid may have a high activity, whereas enzymes like
aconitase, isocitric dehydrogenase, malic dehydrogenase
which catalyse the degradation of citric acid may demonstrate
a low activity.

When attempting to correlate metabolite accumulation

with an in vitro estimation of enzyme activity it is assumed

24
that the enzyme is the limiting factor and an increase or a
decrease in its activity will affect the concentration of
the metabolite. This assumption is not always valid. Laties
(1964), for example, demonstrated that Krebs cycle enzymes
were present in young potato tubers in a latent state and
that the cycle began to Operate upon the aging of the tuber.
Standardized procedures for the extraction of the enzyme and
their assay is another inherent difficulty in such a survey.
They do not ensure optimal activity for the various possible
enzymic multiple forms. The various cofactors, inhibitors,
activators do not necessarily respond equally well to the
standardized treatment, and may result in deviation from the
optimum. The activity of the enzyme under physiological
conditions may differ from that under assay conditions. Al-
though these reservations are not uncommon to other in vitro
experiments, The validity of the conclusions of the in
yitrg studies were checked with in—viyg experiments in this
study.

Comparison of Enzyme Activity in
Various Fruits

Two groups of fruits were assayed for their enzyme
activity.

1. Citric accumulators included the fruits of

25
tomato, strawberry, Bartlett pear, and a
few species from the citrus family like
orange, tangerine, grapefruit, and lemon
fruits.
Non-citric accumulators included the fruit
of apples and sweet lime which are malic
accumulators and Naval orange flavedo which
is an oxalate accumulator.
Five enzymes of the Krebs cycle were assayed: acon—
itase, isocitric dehydrogenase (TPN dependent), isocitric
dehydrogenase (DPN dependent), malic dehydrogenase, and
citric synthase. From the glyoxylate cycle isocitritase
was assayed. All the results are averages of three experi—
ments, and the maximum deviation is 115%.

Aconitase

 

Apples and sweet limes (non-citric accumulators)
and oranges and strawberries (citric accumulators) were
assayed for their aconitase activity. The results are sum—
marized in Figure 2 and Table I and indicate no difference
between citric and malic accumulating fruits.

Isocitric dehydrogenase (TPN dependent)

 

Apples (malic accumulator) and oranges and straw—

26

 

 

 

 

 

0.1110 ' V
. O
0.100 _
1 0 e
II
‘3 A '
5
‘8
8
'3
.3
O
g 0.050.
3
A——-—A eyple
Q—Q etmbem
O —O crane.
/, .-—-——. sweet line
0.000 A 1
0 10 20
Time (min. )

Figure 2. Aconiteee ectivity in some fruits.

Reaction mixture: 0.1 m1 easy-e. 225 In eole poteeeiu
phoephete pH 7.4. 75 p mole citrate pH 7.“.
Tot. vol. 3 l1.

27

TABLE I

ACONITASE SPECIFIC ACTIVITY

 

 

Fruit Specific activity*
Apple 0.070
Strawberry 0.050
Orange 0.058
Sweet line 0.060

 

*Specific activity is defined as: increase in absorbance
at 240 mu per gram fresh tissue per minute.

TABLE II

ISOCITRIC DEHYDROGENASE (TPN DEPENDENT) SPECIFIC ACTIVITY

 

 

Fruit Specific activity*
Apple 0.14
Strawberry 0.09
Orange 0.12

 

*Specific activity is defined as: increase in absorbance
at 340 mu per gram fresh tissue per minute.

28
berries (citric accumulators) were assayed for isocitric
dehydrogenase activity and the results are summarized in
Figure 3 and Table II. The activity of TPN dependent iso-
citric dehydrogenase is similar in citric and malic accumu-
lators.

Isocitric dehydrogenase (DPN dependent)

 

 

Apples, strawberries, and oranges were assayed for
the activity of DPN dependent isocitric dehydrogenase. No
activity was detected, even though the assay was performed
at two pH levels (7 and 9). This is supported by Bogin
(1966) who found only TPN dependent isocitric dehydrogenase
activity in lemon and sweet lime. The reaction mixture
'was: 20 umole isocitric acid, 100 umole potassium phosphate
pH 7.0 or 9.0, 10 umole magnesium nitrate, 0.2 ml enzyme.
Total volume 3.0 ml.

Malic dehydrogenase

 

Apples, oranges, sweet lines, and lemons were assayed
for malic dehydrogenase activity. Again no difference in
activity could be demonstrated between the two groups (Figure
4). It is noteworthy that in terms of TPN reduction malic
dehydrogenase activity is at least ten times higher than that

of any other dehydrogenase tested in this work.

29

 

 

 

 

 

 

0.150
A
'1
O
O
I. 0.100”
. A
.3 0
3 0
3
‘fi
0
C
'3
3
O
C
3
0 0.0504.-
.5
O O
A—A m1.
O—-——O orange
O—O etm‘berry
0.000 '
O 5 10
Time (aim)
Figure 3. Ieocitric delvdreeeueee (m dependent ) activity of some

{mitle

Reection mixture: 100 uncle potassium phosphate pH 7.0.30 mole

ieocitrete. 10 mole negneIiun nitrate. 0.5 mole TPN.
0.2 ml 03”.. Tate 701. 3.0 do

30

 

 

 

 

0.600
A
s
A
O
I
1 o A
'3 0.u00 -
g .
g 0
e
0
5 r
.0
a
a D t
a A\
.‘3
3 r
: OeZOO ?" D
3
C
A—A apple
CI——-D lemon
O—O ounce
0.000 l '
O 2 it
Time (11111.)

Figure 4. Malic delvdrogenaee activity of acne fruite.

Reaction mixture: 1 mole oxalacetate pfi.7.l+. 0.1 mole DEB.
100 mole potaeeiun phcephate pH 7.11, 0.02 ml enzyme.
Tot. vol. 3.0 ml.

31

Citric synthase

Preliminary experiments indicated that citric synthase
activity unlike the rest of the enzymes in this survey is not
identical in all fruits. Since this enzyme may be a key fac-
tor in citric acid biosynthesis, a rather extensive survey,
involving ten tissues was conducted in order to see whether
a correlation existed between citric synthase activity and
citric acid accumulation. From the group of citric accumu—
lators the following fruits were examined: green tomatoes,
red and green strawberries, Bartlett pears (which accumulate
equally well citric and malic acids) and from the citrus
family the juice and juice vesicles of orange, lemon, and
tangerine. The other group of fruits included the malic
accumulators, apples and sweet limes and an oxalate accumu—
lator — the flavedo of the naval orange. A broad spectrum
of activities could be demonstrated both on the basis of
miligram acetone powder and gram fresh tissue (Figure 5,
Table III) .

All the fruits of the citrus family exhibited a high
citric synthase activity, regardless of the acid which they
accumulated. Thus the orange, grapefruit, tangerine, and

lemon (all citric accumulators), the sweet lime (a malic

32

 

 

 

 

 

 

 

 

0.060
A—A apple D
D — D lemon 0
O 0 orange
O O strawberry
. . sweet line
‘..__‘ tomato
1
a 0 0&0 D
3 . O
n
2 U
a
o C)
3
'3
3
'3 A
3
3 e
g 0.020 _
3 I
3
s . ‘
"I
/ ‘
" A
0.000 ° 0 Q C C
0 3 6 9
Time (min. )

Figure 5. Citric synthase activity of some fruits.

Reaction mixture: malic acid 10 mole. DPN 0.3 mole.potassiun
phosphate pH 8.0 75 mole. acetyl CoA 0.2 mole.0.l ml same.
Tot. vol. 1.0 I1.

33

TABLE III

CITRIC SYNTHASE SPECIFIC ACTIVITY

 

 

Fruit Specific activity*
Tangerine 0.090
Orange 0.070
Lemon 0.070
Grapefruit 0.060
Tomato 0.060
Strawberry 0.004
Bartlett pear 0.050
Orange flavedo 0.240
Sweet lime 0.055
Apple 0.020

 

*Specific activity is defined as increase in absorbance at
340 mu per gram fresh tissue per minute.

34

accumulator), and the orange flavedo (an oxalate accumulator)
had a high activity. Tomato (a citric accumulator) and apple
(a malic accumulator) demonstrated an intermediate activity.
The Bartlett pear (malic and citric accumulator) had a medium
activity, whereas the strawberry which is a citric accumulator
exhibited a very low activity regardless of the degree of
ripeness.
Dialysis

To remove all possible low molecular weight activators
or inhibitors the enzyme extracts from oranges and apples
were dialyzed and retested. As can be seen in Figure 6 dial-
ysis reduced the citric synthase activity in both oranges and
apples, but the ratio of activities remained unchanged, 3.6
and 3.8 for undiahfized orange and apple and for dialfized
orange and apple, respectively. Apparently, no dialfizable
activator or inhibitor is associated with high citrate syn—
thase activity of the orange over that of the apple.

Isocitritase

 

Two different reaction mixtures and two pH values as
well as different reagents were tried in the assay for iso—
citritase activity. Isocitritase is the key enzyme of the

glyoxylate pathway but no activity was observed in the tissue

35

 

 

 

 

0.060
El ——-E1 undialysed apple extract
I—I dialyzed apple "
O—O undialysed orange '
.— . dialyzed orange '
0
d
's
a. O
' 0.040.. O
O
3
n
4’
e
0 O
3 .
O
O
'8
.9
e
° CI
.3 I, . r
I
O .
I
a
0.000. .1 i
0 5 10
Time (min. )

ngre 6. Effect of dialysis on citric synthase activity of apples
and oranges.

Reaction mixture: malic acid 10 panels. In 0.3 pmole.potassium

phosphate pH 8.0 75 mole. acetyl 00A 0.2 mole. 0.1 ml enzyme.
Tot. vol. 1.0 ml.

36
examined. This is in line with other observations (Carpen—
ter and Beevers, 1959). The two reaction mixtures were as
follows: 1) 200 umoles potassium phosphate pH 7.6, 15

umoles MgSO 6 umoles cysteine HCl, 24 umoles isocitrate,

4.
water to 2.8 ml and 0.2 m1 enzyme. The reaction was termi—
nated by the addition of trichloroacetic acid. 2.4 dinitro-
phenylhydrazine was the color reagent. 2) Twenty umoles
cysteine HC1, 180 umoles semicarbazide, 50 umoles MgSO4,
600 umoles, 20 umoles sodium isocitrate, 200 umoles potas—
sium phosphate pH 6.0, water added to 2.8 ml and 0.2 ml
enzyme. The semicrbazones were followed at 252 mu.

In conclusion, the comparison of enzymatic activi-
ties in various fruits which are citric or malic accumulators
did not substantiate the theory that enzymic imbalance in
the TCA cycle is the cause for citric acid accumulation.

The activity of aconitase, isocitric dehydrogenase (DPN and
TPN dependent) and malic dehydrogenaseis virtually identical
in malic and citric accumulators. From the standpoint of
citric acid accumulation all these enzymes can be considered
degradative enzymes, that are leading to the consumption of

citric acid.

Citric synthase on the other hand exhibited a wide

37
range of activities, but no correlation could be found be-
tween the activity of this enzyme, and acid accumulation in
fruit.

If any of the accumulated citric acid originates in
the TCA cycle, a source of replenishing the cycle intermedi—
ates biosynthetically related to citric acid must exist.

There are metabolic pathways capable of feeding the cycle

with intermediate compounds. Kornberg (1957) who worked on
the glyoxylate pathway in bacteria, and showed that it can
lead to a net synthesis of malic acid, hypothesized that

this may lead to the replenishment of malate needed for citric
acid accumulation. But a survey conducted by Carpenter and
Beevers (1959) failed to show isocitritase activity in any
plant except for certain germinating seeds. The present study
with isocitritase confirmed Carpenter and Beevers' observation
that fruit tissues are devoid of isocitritase activity.

Reversal of the isocitric dehydrogenase reaction
could replenish isocitric acid into the cycle. The incor-

poration of CO to isocitric acid was reported by Vennesland

2
and Conn (1955). By using the oxidation of TPNH as the sole

criteria of CO2 addition to (at-ketoglutarate by orange acetone

powder; no formation of isocitric acid could be demonstrated.

38
The decarboxylation of isocitric acid has been established
equally well in all fruits which were examined. ci-keto—
glutaric acid is a very unlikely source for citric acid
accumulation since citric acid is the only known source for

its formation.

Radioactive IsotopeExperiments

O ' C I I I 0
Since enzyme actiVity was assayed pp Vitro it was

 

of interest to correlate it with ip yiyg experiments. In—
jection of radioactive precursors seemed to answer this
requirement. In injecting small quantities of radioactive
compounds with high specific activity, it was assumed that
the tagged compound would mix with the same compound in the
.fruit and cause a minimal interference with the metabolism
of the fruit. This method has inherent difficulties. A
radioactive precursor administered from outside the cell may
reach the internal pools and biosynthetic sites at a differ-
ent rate than the native precursor. This is especially true

when intermediates are being injected. In vivo, enzyme com—

 

plexes may catalyse a successive transformation of a compound,
without allowing any leakage of intermediates to the existing

pool. It is quite conceivable that when an intermediate is

administered from outside the cell it may participate in other

39
reactions before reaching the active site. The side reactions
may mask the main reaction in which this intermediate par—
ticipates lg yiyg.

Incorporation p£_Acetate 2—14C

 

 

Labeled acetate 2—14C was injected into tomatoes and
apples (Tables IV and V). During the first three hours the
label was mainly incorporated into citric acid in both fruits.
However, 24 hours after the injection, citrate remained in
the tomato as the main labeled acid, while malic acid was
the main labeled acid in the apple.

It may be concluded that the initial rate of citric
acid synthesis is very similar in tomato and apple regardless
of the acid which is accumulated. After longer incubation
time, the 14C distribution shows similarity to the acid pat—
tern in the given fruit. In the apple the ratio of 14C in
the citric acid to the 14C in the other acids shows a decline
from 1:1.1 half an hour after injection to 1:23 twenty-four
hours after injection. The inverse happens to malic acid
which increases from a ratio of 1:30 half an hour after in—
jection to 1:3.5 twenty-four hours after injection (Table V).
There are at least two possible explanations for this ob—

servation. One is the conversion of citrate to malate

40

TABLE IV

INCORPORATION OF l4 14

C OF ACETATE 2- C INTO THE ORGANIC
ACIDS OF DETACHED GREEN TOMATO FRUITS*

 

 

 

 

 

Time Counts per minute
after per fruit Citric Malic
injection Malic Citric Total /rest /rest
20 min 700 5650 11700 1:1 1:16
40 min 700 5400 9350 1:0.7 1:12
180 min 550 7000 11000 1:0.6 1:19
18 hr 1500 17000 20000 1:0.2 1:12
24 hr 11650 38550 57900 1:0.5 1:4
*Each time interval represents one fruit.
TABLE V
14 14
INCORPORATION OF C OF ACETATE 2— C INTO THE
ORGANIC ACIDS OF DETACHED PRECLIMACTERIC
NORTHERN SPY APPLE FRUITS*
Time Counts per minute
after per fruit
injection Citric Malic
(hours) Malic Citric Total /rest /rest
0.5** 300 4300 9300 1:1.1 1:30
1.0 650 7700 14100 1:0.8 1:20
2.5 3400 4300 19200 1:3.5 1:4.5
24.0** 6800 1300 31000 1:23 1:3.5

 

* Each time interval represents one fruit.

** Data represent two single fruit experiments.

41
through the TCA cycle; in the tomato only part of the citrate
would be further processed in the cycle, while the rest would
be removed from it; in the apple practically all of the
citrate would be converted to malate. A second speculation
would involve the slow and steady condensation of malate
(Thunberg reaction) while citrate is accumulated (tomato) or
degraded (apple).

These results support the observation from the en-
zyme activity study in that the Krebs cycle enzymes, although
possibly participating in the synthesis of citric acid and
malic acid, do not control their accumulation and do not
determine the acid profile.

Another theory which has been advanced to explain
citric acid accumulation is the existence of an inhibitor
which blocks one of the citric degrading enzymes in the TCA
cycle, and thereby causes citric acid accumulation.

The accumulation of radioactive malic acid in tomato
from labeled acetate refutes this theory indirectly. If an
inhibitor had been present there would not have been any
accumulation of labeled malic acid.

Incorporation pf Uniformly Labeled Glucose -14C

 

 

Uniformly labeled glucose -14C was the second precur-

42
sor that was injected to both apples and tomatoes. The re—
sults appear in Table VI. While the results do generally
support our observation with acetate 2—14C there are also
marked differences.

In the apple, during the first 2 1/2 hours, citric
acid accounts for 65%.pf the total acid radioactivity and
only later, at 24 hours, does malic acid take over (Table VI).
In the tomato, however, the picture becomes more complicated
by the appearance of an unknown radioactive peak which is
the major peak during the first few hours after infiltration
(Figure 7). It later declines in activity while the activ-
ity of citric acid and malic acid goes up (Table VII). In
accordance with the previous results, 24 hours after the
glucose injection in the tomato the highest radioactive
count is found in the citric acid fraction. By injecting
simultaneously glucose-UL - 14C and phosphate — 32F it was
shown (Figure 8) that this unknown compound is phosphory—
lated.Incorporation of 14C in the unknown compound appearing
early after injection and then declining with a concurrent
increase in radioactivity in citric and malic acidsfwsuggest
that this unknown compound is a precursor of citric acid.

An attempt was made to test whether the phosphorylated

43

TABLE VI

14

INCORPORATION OF

C OF GLUCOSE U.L.
ORGANIC ACIDSOF DETACHED APPLE FRUITS

14

C INTO

 

Counts per minute

 

Time per fruit Citric Malic
(hours) Malic Citric Total /rest /rest
0.5 0 2300 3500 1:0.5 —
1.0 50 2800 5250 1:0.9 1:100
2.5 - 600 1500 1:1.5 -
29 1800 1200 4350 1:2.5 1:1.4

 

44

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47

compound might be a precursor of citric acid. Double—labeled
unknown 14C and 32P which was isolated by column chromato-
graphy from tomato fruit was injected into another tomato.
After an hour the analysis failed to show any radioactivity
in the citric acid, but some radioactivity appeared in the
phosphoric acid fraction. These results suggest that this
compound was probably hydrolysed in the cells of the tomato.
Hence, the role of this unknown compound is still obscure.
It has not been shown that it is a precursor of citric acid.
but because of its hydrolysis it can not definitely be ruled
out as a precursor. This compound was not found in apple.

Bakowski e£_al, (1964), and Markakis and Embs (1964)
working on snap beans and strawberries respectively, demon-
strated an acidic compound that appeared in the elution
pattern on the shoulder of the citric acid peak. This peak
coincides with the unknown in the present study.

Markakis and Embs (1964), however, demonstrated that
their unknown compound contained radioactivity after 24 hours
when radioactive fructose was administered to strawberries
while in the present investigation no traces of radioactivity
were found in the unknown after 24 hours. This discrepancy

may be due to different tissues employed,

48

Homogenate

 

In order to learn more about this unknown compound
and its relationship to citric acid synthesis, uniformly
labeled glucose —14C as well as glucose -l—14C and glucose
—6-14C were added to tomato homogenate. When ATP, DPN, and
Mg were added, the unknown became labeled. All attempts to
label the citric acid fraction failed. (Table VIII). The
formation of the unknown was repressed by addition of boiled
tomato extract. On the basis of the present knowledge it is

difficult to speculate on the nature of this repression.

Addition of NaHCO , in hopes that CO

3 fixation may result

2
in formation of citric acid, failed to change the 14C dis—
tribution in the acid profile. Addition of sucrose or its
withdrawal from the homogenized media did not have an influ—
ence on the 14C distribution and did not affect appreciably
the reate of incorporation. It may be concluded that the
degree of integrity of the mitochondria is of no importance
in the biosynthesis of the unknown. The fact that glucose
-l-14C and —6-14C served as precursors rules out the pentose
shunt as a possible source of this unknown and almost rules

out decarboxylation as a step for its formation.

It must be emphasized that these experiments also

INCORPORATION OF 14

49

TABLE VIII

14

C FROM GLUCOSE— C INTO THE ORGANIC

ACIDS BY TOMATO HOMOGENATE

 

Homogenate with the
following additives

Counts per minute per
three gram tomato
Glucose -Malic Citric Unknown

 

*Sucrose

*Suc., ATP, DPN, TPN,

*Suc., ATP, DPN, TPN,

*ATP, DPN, TPN, Mg
*Suc., ATP, DPN, TPN,
*Suc., ATP, DPN, Mg
**Suc., ATP, DPN, TPN,

**Suc., ATP, DPN, TPN,
boiled tomato

**Suc., ATP, DPN, TPN,

NaHCO3

**Suc., ATP, DPN, TPN,

M9
M9

M9

Mg
Mg,

Mg,

Mg,

NaHCO , boiled tomato

3

UL-14C 0 0 200
UL-14C 0 0 4500
6-14C 0 0 3500
1—14C 0 0 3000
1-14C 0 0 4500
1—14C 0 0 4500
UL-14C 0 0 4300
UL-14C 0 0 1400
UL-14C 0 0 6000
UL—14C 0 0 1400

 

Homogenate plus additives incubated for three hours.

Homogenate is composed of 3 g tomato, 300 umole potassium
phosphate pH 7.5 homogenized for 20 seconds, the final pH

is 7.1.
*Final volume 10 ml.

**Final volume 20 ml.

Boiled tomato consists of 5 g tomato homogenized in 250
umole potassium phosphate pH 7.5, boiled for 5 minutes.

Total volume 10 ml.

Glucose l uC

ATP 40 umole
TPN 5 umole
DPN 5 umole

Mg(NO ) 10 umole

NaHCO3 lOO umole

50
failed to clarify the role of the unknown as a precursor of
citric acid. This is especially so since attempts to achieve
citric acid biosynthesis by tomato cell—free extract from

glucose failed,

Attempts to Identify the Unknown

Double Label Experiments

 

Glucose -l—l4C and phosphoric acid -32P were injected
simultaneously into the tomato. The carbon peak and the
phosphate peak coincided indicating that this compound con—
tains both 14C and 32P.

Column Chromatography

The unknown compound is an acid. It is not adsorbed
by Dowex—SO, a cation exchange and is adsorbed by Dowex-l
which is an anion exchange.. Its position on the elution
pattern between citric and phosphoric acid indicates the

relatively strong anionic nature of this acid.

Paper Chromatography_

 

The unknown compound was subjected to paper chromato-
graphy with known compounds (Table X). The Rf of the un-
known differed from all others, thus eliminating few possi-

bilities but yet leaving open the question of its identity.

51

TABLE IX

2
INCORPORATION OF 14C OF GLUCOSE U.Lm -14C AND PO4 -3 P

INTO ORGANIC ACIDSOF DETACHED GREEN TOMATO FRUITS

 

Counts per minute per fruit

Time
after Malic Citric Unknown Phosphoric Total
injection C P C P C P C P C P

 

2.5 hr 7000 D 11700 5200 6200 47500 9 500000 35800 552000

4 hr 15000 0 20200 24500 0 92500 '0 34500 52500 484700

 

TABLE X

Rf VALUES OF SOME PHOSPHORYLATED COMPOUNDS

‘—
-_—

 

Compound Rf
Glyceraldehyde phosphate 0.23
3-phospho glyceric acid 0.19
Phospho enol pyruvic acid 0.30
Adenosine 5' mono phosphate 0.03
Adenosine 5‘ diphophate 0.02
Inosine 5' mono phosphate 0.04
Fructose -1— phosphate 0.05
Glucose —1 phosphate 0.05
Unknown 0.13

 

A twice ascending chromatography was employed. The paper
used was Whatman No. l, and the solvent was ethyl-methyl—
ketone: water saturated butanol: propanol: formic acid
45:25: 5:20 (V/V)

52

Compartmentation

 

The enzymic survey and the precursor studies did not
support the theory that overproduction of citrate is the
cause of citrate accumulation. The data in Tables V and VI,
however, are compatible with, although not a proof for, the
theory of compartmentation. According to this assumption,
citric acid in apple, although biosynthesized both from glu—
cose and acetate in the first few hours, does not accumulate
but is metabolized directly into malic acid which accumulates
in stable pools. The other possibility is that citric acid
is metabolized to CO2 and H20 and malic acid arises via a
different mechanism. This assumption seems to explain the
slowness in the appearance of malic acid in apples when glu—
cose is the precursor.

According to the compartmentation theory the accumu-
lation of an acid is a result of active transport from the
site for synthesis into a passive pool. This rather than
excess of enzyme prompts further synthesis of this acid.

Bennet—Clark and Bexon (1943) have experimental evi-

dence that individual acids may be constrained inside the

vacuole so that an apparent diffusion gradient is maintained

53
between the vacuole and the cytoplasm. Compartmentation of
cytOplasm has been invoked by many authors (Porter and May,
1955; Steward, Bidwell and Yemm, 1958; Cowie and McClure,
1959). Beevers and his collaborators (Harley and Beevers,
1963; MacLennan gt 21:! 1963; Lips and Beevers, 1966 a, b;
Lips, Steer and Beevers, 1966) demonstrated compartmentation
in some plant tissues.

Actual compartmentation of acids has been shown by
Schneider §£_§1,. (1956). They showed that in fluoroacetate
poisoning, which causes citrate accumulation, most of the
citric acid is found in the mitochondria of rat liver.

A serious drawback in compartmentation studies is
that the most important compartment, namely the vacuole, is
mixed with the cytoplasm, during the isolation of particles.
It is also true that all possible and hypothetical compart—
ments in the cytoplasm will be mixed, Nevertheless, since
particles, especially the mitochondria, seem to be the loca—
tion for the synthesis and degradation of organic acids,
this examination seemed to be of merit.

Studies were made to measure the 14C incorporation
into particles and supernatant of both the tomato and apple

at several intervals (Table X) after injecting them with

54
2—14C acetate. The isolation of mitochondria was done at
OO—ZOC to reduce leaking and further metabolism of the acid
by the mitochondria stem.

While 5% of the radioactivity was confined in part-
icles of tomatoes, 30%.was found in the particles of the
apple, after the administration of the 2—14C acetate. When
the incubation time was extended, particles were depleted
from radioactivity in both fruit. Citric acid was the only
labeled acid found in the particles from apples.

This may suggest that although citric acid is the
first acid to be biosynthesized by apple from acetate, it

is retained in the particles and metabolized there. It may

be either converted to malic acid and then released to the

cytOplasm or completely oxidized to H 0 and CO and the malic

2 2
acid which is accumulated by the apple may be synthesized by

a different biochemical route.
These results may point to another factor in acid
accumulation, namely, membrane permeability and compartment—

ation.

55

TABLE XI
l4 l4
INCORPORATION OF C FROM ACETATE -2- C INTO THE
ORGANIC ACIDS OF PARTICULATE AND SUPERNATANT
FRACTIONS OF APPLE AND TOMATO FRUITS

 

 

Time Counts per minute Ratio
after' particles
Fruit injection Particles Supernatant /super.
(Hours)
Apple 0.5 500 1000 1:2
4.0 120 700 1:7
15.0 50 600 1:12
24.0 30 530 1:17
Tomato 1.0 270 5000 1:18
9.0 150 4000 1:29
24.0 60 5100 1:85

 

V. SUMMARY AND CONCLUSIONS

1. The acetone powders of the orange, lemon, tangerine,
grapefruit, strawberry, and tomato fruits (all citric acid
accumulators), the apple and sweet lime fruits (malic acid
accumulators), the orange flavedo (an oxalate accumulator)
and the Bartlett pear (a fruit with high malic and citric
acid content) were assayed for the activitnsof the following
enzymes: aconitase, isocitric dehydrogenase (TPN dependent),
isocitric dehydrogenase (DPN dependent), malic dehydrogenase,
citric synthase, and isocititase.

2. The activity of aconitase, isocitric dehydrogenase,
malic dehydrogenase, and citric synthase was Observed in all
of the tissues studied. The activities of isocitric dehydro-
genase (DPN dependent) and isocitritase were not detected in
these tissues. Aconitase, isocitric dehydrogenase, and malic
dehydrogenase showed activities which did not differ consid-
erably from one fruit to another. Citric synthase varied
markedly from fruit to fruit, but no correlation between

citric acid content and this activity was apparent.

56

57

3. Acetate 2—14C was injected to detached green tomatoes

and apples. Half an hour after injection the incorporated
C was associated with citric acid in both fruits.

After 24 hours citric acid remained the most labeled
acid in the tomato while malic acid was most highly labeled
in the apple.

4. Glucose-U.L.l§C, was injected into detached green
tomatoes and apples. In the apple, citrate was highly labeled
aeéd one hour after injection but malic acid became the dom-
inant labeled acid after 24 hours. In the tomato an unknown
compound incorporated a major portion of the 14C from glucose
within 30 minutes after injection and declined with time with
a concurrent increase in labeled citric and malic acids and
after 24 hours most of the label was in the citric acid frac—
tion and none in the unknown.

5. A double label experiment with 14C and 32F indicated
that the unknown acid must be a phosphorylated compound.
Paper chromatography revealed that the unknown was not one
of the following compounds: glyceraldehyde-phosphate, 3—
phosphoglyceric acid, phosphoenolpyruvic acid, adenosine
monophosphate, adenosine diphosphate, inosinemonophosphate,

fructose-l—phosphate, glucose -1 phosphate and glucose - 6

58
phosphate. Homogenate studies established the dependence of
this unknown on ATP for its biosynthesis. The label of glu-
cose-U.L.-14C as well as glucose-l-14C and glucose—6-14C was
found in this compound, eliminating decarboxylation as a
step in its biosynthesis. In addition acetate-2—14C did not
serve as a precursor.

6. Acetate 2—14C was fed to apples and tomatoestIhe
distribution of radioactivity between particles precipitated
at 22,000 x g and the supernatant fraction was compared. In
the apple, 33% of the organic acid radioactivity was found
in the particles compared with only 5% in the tomato. Citric
acid was the only labeled acid in the apple particles.

7. It is concluded that the organic acid profile in the
fruit tissues examined does not correlate with the enzyme
activity profile. Labeled precursor experiments corroborate
the enzyme survey finding. Finally the intracellular distri-
bution of newly synthesized organic acids indicates the pos—

sibility of compartmentation as a factor in acid accumulation

in fruits.

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