Title: Biochemical basis of acidity in citrus fruits
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Title: Biochemical basis of acidity in citrus fruits
Physical Description: 64 leaves : ill. ; 28 cm.
Language: English
Creator: Buslig, Béla Stephen
Publication Date: 1970
Copyright Date: 1970
 Subjects
Subject: Citrus fruits   ( lcsh )
Fruit Crops thesis Ph. D
Dissertations, Academic -- Fruit Crops -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1970.
Bibliography: Includes bibliographical references (leaves 61-63).
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Be'la Stephen Buslig.
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 000414601
oclc - 37760563
notis - ACG1781

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BIOCHEMICAL BASIS OF ACIDITY IN CITRUS FRUITS














By
BE'LA STEPHEN BUSLIG













A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA

1970













ACKNOWLEDGMENTS


The author expresses sincere appreciation to the members of

his supervisory committee for their help throughout these series of

investigations. To Drs. R. H. Biggs, chairman, and J. A. Attaway,

co-chairman of the committee, for their infinite patience and aid

during various parts of these studies, are due special thanks.

Further thanks are in order to Dr. A. H. Krezdorn, who was

instrumental in initiating the author's studies in the Department of

Fruit Crops, and to Drs. W. F. Newhall and D. S. Anthony for valuable

discussions.

The author's sincere gratitude is extended to Mrs. Fay Ball

for the expert typing and for her patience during the preparation of

the manuscript.

Last, but not the least, very special thanks are due to the

author's wife, Bertha, who provided constant encouragement throughout

the entire tenure of graduate studies.


ii














TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS........................... ............ ii

LIST OF TABLES.......................... ............. v

LIST OF FIGURES ............................................ vi

ABSTRACT ..................... .................... vii

INTRODUCTION................................... .

LITERATURE REVIEW ............................ ............ 2

MATERIALS AND METHODS ......................................... 11

Plant materials......................................... .. 11
Sampling ................................ .... .. ... 11
Preparation of tissue extracts ............................. 11
Isolation of mitochondria.............................. 13
Measurement of mitochondrial respiration................. 13
Determination of protein .................................. 14
Determination of phosphorylation.......................... 14
Determination of enzymic activities...................... 14
Measurement of ATP and ADP concentrations.................. 15
Measurement of whole fruit respiration.................... 15
Acidity measurements ........................ ........ 16
Incorporation of 14C02 into whole peeled citrus fruit...... 16
Radioactivity measurements................................. 16

RESULTS .......................................... .............. 18

Titratable acidity and the respiratory activity of
various citrus fruits................................. 18
Respiratory activity of isolated fruit mitochondria........ 23
Enzymic activities of fruit pulp during growth............ 32
Concentrations of ATP and ADP in fruit pulp............... 41
Metabolism of 14C-citrate and 14C-pyruvate by
isolated mitochondria and whole juice sacs............... 41
Dark fixation of 14C02 by whole peeled fruit............. 49
The effect of arsenate on mitochondrial activity
of Valencia oranges............................. ... 49


iii










)iSC SSIi O* 5
J. SCisS I . .. .. .. .. .. .. .. .. .. ............ 52

S IX . . . . . . ....... 60

BIBLIOGRAPHIY

























































iv













LIST OF TABLES


!able Page

1. Experimental materials .......................... 12

Acidity and respiration of mature citrus fruits... 24

3. Respiration of mature citrus fruit mitochondria... 25

4. Concentration of ATP and ADP during development
in Baboon lemon, bittersweet and sour orange.... 46

5. Metabolism of radioactive citrate and pyruvate
by isolated mitochondria. ..................... 47

6. Metabolic activity of whole juice sacs............ 48

7. Incorporation of 14C02 into whole peeled fruit.... 50

8. Respiration of Valencia orange mitochondria....... 51













LIST OF FIGURES

Figure Page

1. The Krebs cycle................................. .... 6

2. Patterns of titratable acid accumulation in
bittersweet and sour oranges and in sour lemon....... 20

3. Respiration of whole fruits during growth............ 22

4. Respiration of bittersweet orange mitochondria........ 27

5. Respiration of sour orange mitochondria............... 29

6. Respiration control ratio changes during
development. ................... . .................. 31

7. Citrate synthetase activity of bittersweet oranges.... 34

8. Citrate synthetase activity of sour oranges........... 36

9. NADP-isocitrate dehydrogenase activity of
bittersweet oranges.................... ............ 38

10. NADP-isocitrate dehydrogenase activity of
sour oranges................................. 40

11. NAD-isocitrate dehydrogenase activity of
bittersweet oranges................................. 43

12. NAD-isocitrate dehydrogenase activity of
sour oranges................................. .... 45









Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy

BIOCHEMICAL BASIS OF ACIDITY IN CITRUS FRUITS

By

Bdla Stephen Buslig

December, 1970

Chairman: Dr. R. H. Biggs
Co-Chairman: Dr. J. A. Attaway
Major Department: Fruit Crops


The metabolism and accumulation of organic acids in citrus fruits,

with emphasis on citric acid, were investigated. The problem was

approached by examining the accumulation patterns of titratable organic

acids in various fruits, while concurrently studying respiration, both

in the entire fruit and in subcellular fractions (mitochondria). The

enzymes citrate synthetase, isocitrate dehydrogenases and isocitritase

were studied. Results indicated better coordination of enzymic activity

and respiration in less acid fruit. Examination of ATP and ADP levels

in fruits of varying acidity indicated the involvement of these

nucleotides in respiratory control and probably active transport of

overproduced citric acid into a separate compartment of the juice sacs,

probably the vacuole. The results obtained from C02 fixation studies by

whole fruit seemed to discount the idea that carboxylation reactions are

significant contributors to acid production. Another experiment con-

cerned with the action of arsenate on respiration indicated that

arsenate acts in competition with phosphate, and not as an uncoupler.







Experimental evidence also points to greater respiratory efficiency in

less acid fruit mitochondria, with better respiratory control by

exogenous ADP. Conclusions drawn from the experiments point to

coordination of enzymic activities as the main pathways in controlling

organic acid accumulation.













INTRODUCTION


The genus Citrus includes the largest single group of economic fruit

producing plants in the world. Among the numerous diverse characteristics

of these fruits is the remarkable variation of acidity within the group,

or even within varieties of a single species. It ranges from 0.06 to

8.00% titratable acids, expressed as citric acid, in the juices of the

different variants.

The metabolism and accumulation of organic acids, especially citric

acid which predominates in most varieties, is therefore of particular im-

portance. An understanding of the basic processes involved could possibly

contribute means to control acidity levels in these fruits. Investigation

of the biochemical pathways leading to the synthesis and degradation of

citric acid, primarily reactions associated with the Krebs cycle and some

of zhe associated reactions, could provide insight into the regulation of

organic acid metabolism. Eventual deciphering of the enzymic mechanisms

and their integrated control could possibly be used to devise ways to

control acid accumulation, which involves the phenomenon of membrane

transport, across both the mitochondrial and the vacuolar membranes.

Accumulation involves transport against a concentration gradient. Energy

relationships obtained during the growth cycle of the fruit integrated

with the enzymological and acidity accumulation data are expected to shed

light on the biochemical basis of citrus fruit acidity.












LITERATURE REVIEW


Changes in organic acids in different citrus fruits were the sub-

ject of the first lines of investigation into acid accumulation. Two

distinct patterns of accumulation were distinguished.

Lemons (Citrus limon (L.) Burm.) followed a pattern of continuous

accumulation during the growth of the fruit. The increase was greatest

during the early growth of the fruit. It continued at a slower rate

while the fruit was in a logarithmic growth phase, until the end of the

cycle. The increase in titratable acidity was continuous until maturity,




Abbreviations and terms used in the text are given below: ADP
(adenosine diphosphate); ATP (adenosine triphosphate); PEP (phosphoenol-
pyruvate); dimethyl-POPOP (l,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene);
PPO (2,5-diphenyl-oxazole); AcSCoA (acetyl-coenzyme A); CoASH (coenzyme A);
NAD (nicotinamide-adenine-dinucleotide, oxidized form); NADH (nicotinamide-
adenine-dinucleotide, reduced form); NADP (nicotinamide-adenine-dinucleotide
phosphate, oxidized form); NADPH (nicotinamide-adenine-dinucleotide phos-
phate, reduced form); TRIS (tris-(hydroxymethyl)-aminomethane); MOPS (mor-
pholine-propanesulfonic acid); EDTA (ethylene diamine tetraacetic acid);
DMSO (dimethyl-sulfoxide); RCR (respiratory control ratio); P/O (respira-
tory phosphate:oxygen ratio); BSA (bovine serum albumin); PVP-40 (polyvinyl-
pirrolidone, average molecular weight 40,000); ENZYMES: the trivial name
is used, they are identified here by the International Union of Biochemis-
try Enzyme Commission adopted name and number: adenylate kinase (ATP:AMP
phosphotransferase) (EC 2.7.4.3); aconitase (citrate (isocitrate)-hydro-
lyase) (EC4.1.2.3); citrate synthetase (citrate-oxaoacetate-lyase (CoA-
acetylating) (EC 4.1.3.7)); citrate lyase (citrate-oxaloacetate-lyase)
(EC 4.1.3.6); citrate cleavage enzyme (ATP: citrate oxaloacetate-lyase
(CoA-acetylating and ATP dephosphorylating) (EC 4.1.3.8)); NAD-isocitrate
dehydrogenase (Ls-isocitrate: NAD oxidoreductase (decarboxylating) (EC
1.1.1.41); NADP-isocitrate dehydrogenase (Ls-isocitrate: NADP oxidore-
ductase (decarboxylating) (EC 1.1.1.42)); isocitritase (Ls-isocitrate
glyoxylate lyase) (EC 4.1.3.1); PEP carboxylase (Orthophosphate:oxaloace-
tate carboxy-lyase (Phosphorylating) (EC 4.1.1.31)); PEP carboxykinase
(ATP:oxaloacetate carboxy-lyase(transphosphorylating) (EC 4.1.1.32)).








with the apparent dilution effect caused by the increasing fruit volume

lagging behind the production (Bartholomew, 1923). A similar pattern was

observed in a species termed C. acida (probably a lemon) by Sekhara Varma

and Ramakrishnan (1956) in their investigation of sources of acid for-

mation in these plants.

In oranges (C. sinensis (L.) Osbeck) the pattern of acidity accumu-

lation was different. There was an initial rapid rise of titratable

acids, followed by a long, gradual decline with increasing size, mainly

due to the dilution effect of the increased water content of the fruit.

At the onset of maturity this decline was hastened by a net loss of total

acids (Sinclair and Ramsey, 1944; Bain, 1958; Rasmussen, 1964; Ting and

Vines, 1966).

In grapefruit (. paradise Macf.) a pattern comparable to that of

oranges was observed (Harding and Fisher, 1945; Ting and Vines, 1966).

Generally, it is believed that accumulation of organic acids is

caused by overproduction of a specific acid which cannot be efficiently

metabolized due to a partial metabolic block or enzymic defect, possibly

genetically controlled within the fruit.

The major organic acid found in citrus fruits, with a few excep-

tions, is citric acid. In most instances it is present in excess of 70%

of the total titrable acidity (Erickson, 1968). In addition to citric

acid, malic, succinic, fumaric and quinic acids can be found in appreci-

able quantities in the juice of the various fruits (Ting and Deszyck, 1959;

Clements, 1964 a,b).

All the major acids in the juice, with the exception of quinic,

belong to a group which are interconvertiable through a series of react-

ions known as the tricarboxylic, citric acid or Krebs cycle (Krebs and








Johnson, 1937). Initially, the reactions of the Krebs cycle were deduced

from work with animal tissues. Later the enzymes and reactions involved

were found to exist in all plants examined (see Beevers, 1961; Beevers,

Stiller and Butt, 1966). Figure 1 shows the series of interconversions

occurring in the Krebs cycle.

The first reaction in the Krebs cycle involves the condensation of

AcS-CoA and a component of the cycle, oxaloacetate, to form citrate.

Citrate is further metabolized by the subsequent enzymes of the cycle.

The only known way citrate can be synthesized is by the enzyme citrate

synthetase. Degradation of citrate can be effected by several enzymes

(Mahler and Cordes, 1966), namely aconitase, followed by isocitrate de-

hydrogenases, citrate lyase and citrate cleavage enzyme. The last two

enzymes have not yet been found in citrus. Citrate synthetase was investi-

gated in lemons (Srere and Senkin, 1966; Bogin and Wallace, 1966 b,c) and

in grapefruit (Vines, 1968 a). The characteristics of both enzymes were

similar, resembling the enzymes isolated from animal sources. Aconitase

has not been investigated in citrus, but suggestions for its presence

were obtained from numerous experiments. Isocitrate dehydrogenase exists

in two distinct forms in citrus. One form, specific for NADP as electron

acceptor, is present mainly in the cytoplasm (Buslig and Attaway, 1968 a).

The other enzyme, present in the mitochondria (Buslig, unpublished),

specifically uses NAD as electron acceptor. Other enzymes investigated

in the Krebs cycle of citrus are the malate dehydrogenases (Vines, 1968 b),

which are responsible for the generation of oxaloacetate which is

necessary for the biosynthesis of citrate.




































Fig. 1. The Krebs cycle.








carbohydratess -4 CH3COCO0
pyruvate SCoA Fatty acids
CoASH /
CH3
CO 2 acetyl CoA

21 \ COH-

2(H) -O C --C--OH H 0
SHCH 2
C0 CHCo CH
HO-CH 2 CO IIC
I oxalacetate C-CO2
HCH citrate 2
1 CH2CO2
C2- cis-aconitate
L,- mate H


o \ c-
/CO2 HC-OH
H-C I
II O 2C-CH
,C-H 2
CO- H2
fumarate CO2
there o-Ds- Ls-isocitrate

CO. c02
2H CH C=

H2C Oc SCoA H CH CO2
SC H6H
C2 HCH @ C
succinate U
GTP HCH / \ -ketoglutarate
CoASH c C CoASH
GDP CO2
SsuccinylCoA 21H1









In the experiments of Huffaker and Wallace (1959) the presence of

PEP carboxylase and PEP-carboxykinase was also indicated. These enzymes

also cause the formation of oxaloacetate, and may act as an acceleration

mechanism for the formation of citric acid. The level of these enzymes

was found higher in the more acid lemon fruits.

Examination of nicotinamide-adenine nucleotide redox ratios in

citrus fruits (Bruemmer, 1969) showed an increase of the reduced form of

the NADH/NAD ratio towards maturity, with little change of the NADPH/NADP

ratio. This observation suggested that control of the activity of the

Krebs cycle in citrus was located at the dehydrogenase levels, specifi-

cally at the malate dehydrogenase step.

An enzyme involved in the phosphorylation of adenosine nucleotides,

adenylate kinase, which is related to respiratory energy metabolism, was

also found in sweet and sour lemons. The level was significantly higher

in the sour fruits (Abou-Zamzam and Wallace, 1970).

The major acid in citrus, citric acid, is formed, as mentioned

earlier by the condensation of acetyl-CoA and oxaloacetate. This reaction

is mediated by citrate synthetase which was found exclusively in the

mitochondria in citrus (Srere and Senkin, 1966; Vines, 1968 a). The

location has been found to be in the matrix in liver (Ernster and

Kuylenstierna, 1970). Isocitrate dehydrogenase, as mentioned earlier,

has two forms, the NADP- specific enzyme mainly in the cytoplasm and

the NAD specific enzyme in the mitochondria. These enzymes directly

control the level of citrate in the mitochondria. The activity of citrate

synthetase is regulated by (1) the availability of acetyl-CoA, (2) the

level of oxaloacetate, and (3) the level of ATP in the mitochondrion

(Bogin and Wallace, 1966 c). The supply of pyruvate regulates the









availability of AcS-CoA. The evidence presented by Huffaker and Wallace

(1959) indicates that pyruvate may also regulate the availability of

oxaloacetate by the PEP carboxylation enzymes. Similar results were

obtained by Bogin and co-workers (Bogin and Erickson, 1965; Bogin and

Wallace, 1966 a) with pyruvate-314C and 14C02. The results of these

experiments also gave some indication of the reversal of isocitrate

dehydrogenase as a possible means of synthesizing citrate via isocitrate.

Some of their experiments suggested the blockage of aconitase by citra-

malate as a way of accumulation of citric acids in lemons. Citramalate

presumably would be formed by decarboxylation of parapyruvate utilizing

hydrogenperoxide. In sour lemons the higher catalase activity would

limit this reaction (Clark and Wallace, 1963). A possible in vivo regu-

lation of citrate synthetase by ATP was examined in mature citrus fruit

and a negative correlation was found between acidity levels and ATP con-

centration (Buslig and Attaway, 1969). However, the relation does not

seem to hold true for younger fruit.

Since the enzymes of citric acid biosynthesis and immediate degra-

dation (or reversal of such) are located in the mitochondria (Mahler and

Cordes, 1966) it is obviously the primary site of synthesis. The amount

of citric acid in the mitochondria is limited by the capacity of the

organelle, along with the equilibrium of the Krebs cycle enzymes. Excess

acids are probably transported through the mitochondrial membrane into

the cytoplasm and subsequently into the vacuole (Beevers, et al., 1966).

Spatial separation of the accumulated acids from the enzymes that would

be acting on them predisposes for accumulation. The high H+ concen-

tration in the vacuole is deleterious to most cytoplasmic constituents.










Information on the compartmentation in plant cells is available

from data obtained with Kalanchoe leaves (McLennan, Beevers and Harley,

1963). From this system generalized conclusions may be drawn which are

applicable to acid-accumulating plant organs, indicating the presence of

metabolite pools for the cycle acids, distinct from mitochondria. A

similar conclusion was reached in experiments with maize, where increas-

ing vacuolation was accompanied by less availability of acids to enzymes

in the cytoplams (or mitochondria). In this context it is important to

note that factors controlling movement of solutes from mitochondria to

cytoplasm to vacuole may be important in acid accumulation in plants.

Ranson (1965) cites hypotheses involving active transport into the vacu-

ole of specific acids that are overproduced or alternatively a genetic-

ally controlled system to again actively transport these acids into the

vacuole. In citrus, where varieties of the same species are known to

accumulate acids to different extents, the latter hypothesis is very

attractive.

In commercial practice, some control of citrus acidity is possible

by the use of lead arsenate in post-bloom sprays (Reitz, 1949), but the

mechanism of action of this compound is quite obscure. The effective

moiety is arsenate, which seems to cause more reduction of acids in wet

seasons (Erickson, 1968). Vines and Oberbacher (1965) examined the

effect of arsenate on mitochondria and suggested that the effect is due

to uncoupling of respiration and phosphorylation. However, re-examination

indicates the effect to be competition between arsenate and phosphate ions

during respiration-linked phosphorylation.

Currently, investigation is directed towards the biochemical

events occurring during growth of the citrus fruit. An understanding




10






ot cne biochui..ical events taking place during development, or more

narrowly, citric acid accumulation, and changes in respiratory patterns,

will give a better insight into the biochemical basis of acidity in

citrus fruits.















MATERIALS AND METHODS


Plant Materials

Citrus fruits were obtained from the varieties listed in Table 1,

from the Citrus Experiment Station collection (1, 2) and block plant-

ings (3, 4), and the USDA Orlando Horticultural Station variety

collection (5-9).

Samnling

Fruit of varying physiological ages was collected on the basis of

size. In other cases collection was made in a chronological sequence or

according to season (in mature fruit from the variety collection in

Orlando). It was usually considered desirable to sample and compare

materials from the same trees roughly in the same position to eliminate

genetic and locational differences.

Preparation of tissue extracts

The fruits were homogenized by either Method A, outlined below, or

by Method B. Both methods gave similar results with the substrates

tested.

A. The weighed and peeled fruit was sectioned and grated using a

stainless steel grater into an isolation medium containing 0.25M sucrose,

0.301, mannitol, 0.1M MOPS-KOH buffer, 0.05% BSA, 1% PVP-40, and 0.3%

EDTA, adjusted to pH 7.5. During grating the pH was kept at 7.5 + 0.3

and the temperature below 4 C.

B. The peeled and weighed fruit was solidly frozen by immersing

in liquid nitrogen. After equilibration to the temperature of liquid


11














Table 1. Experimental materials


Sour orange

Bittersweet orange

Marsh grapefruit

Valencia orange

Sampson tangelo

Wekiwa tangelo

Baboon lemon


8. Sweet lemon

9. Succary acidless orange


Description


Citrus aurantium L.

C. aurantium var. Bittersweet

C. paradise Macf.

C. sinensis (L) Osbeck

C. paradise X C. reticulata

C. paradise X. Sampson

C. limon (L.) Burm.

C. limetta Risso

C. sinensis


Type


1.

2.

3.

4.

5.

6.

7.


Pulp at maturity

acidity range
g/1OOg

2.5-4.0

0.3-0.8

0.9-1.5

0.5-1.2

1.0-1.8

0.4-0.8

3.5-4.5

0.05-0.3

0.02-0.1




13





.icrogen the frozen tissue was broken into small pieces and ground to a

fine powder in liquid nitrogen using the lowest possible speed in a pre-

cooled, stainless steel Waring blender. The powdered pulp was left fro-

zen in liquid nitrogen in a Dewar flask until added to isolation medium

in 1:1 (w/v) proportion with constant stirring. The temperature and the

pH were controlled at 0C and 7.5 respectively, the latter by the addition

of 20% KOH containing 0.3M mannitol.

With both methods, the resultant suspension was filtered through a

nylon mesh (Nobel, 1967).

Isolation of mitochondria

Mitochondria were isolated from extracts as prepared above. The

filtered suspension was centrifuged for 20 minutes at 15,000 x g. The

supernatant was used to determine enzymic activities. The pellet was sus-

pended in a wash medium, containing 0.25M sucrose, 0.3M mannitol and 0.1M

MOPS-KOH at pH 7.5. Further fractionation was by differential centrifu-

gation (Buslig and Attaway, 1968).

M-asurc;cnt of mitochondrial respiration

Oxygen uptake of mitochondria was studied by either constant volume

manometric techniques (Umbreit, Burris, and Stauffer, 1964), or polaro-

graphically using the Clark oxygen electrode (Yellow Springs Instruments).

The reaction mixture contained in a final 3 ml volume was 0.25M

sucrose, 0.3M mannitol and 0.05M MOPS at pH 7.5: 100 moles substrate,

70 noles phosphate, 10 pmoles Mg2+, 4 moles NAD, 0.1 pmole coenzyme A,

0.3 imtole thiamine pyrophosphate, 40 pg cytochrome c, 75 pg glucose, 400

hg hcxokinase, and 5-10 mg mitochondrial protein. In case of the Warburg

manometric measurements 15 moles ADP was added 30 minutes after the start

of the experiment to measure the RCR. When respiration was measured by










the oxygen electrode, ADP was added to the mixture by a syringe after the

initial respiratory rate stabilized.

Warburg manometry was performed at 30C, oxygen electrode measure-

ments at 250C.

Determination of protein

Protein in mitochondrial extracts was determined with the Folin

reagent (Sutherland, Cori, Haynes, and Olsen, 1948). The colorimetric

determination was made by measuring the color intensity at 625mp with a

Bausch & Lomb Spectronic 20 spectrophotometer. Bovine serum albumin was

used as standard.

Determination of phosphorylation

Phosphorylation was determined by measuring the glucose-6-phosphate

formed with the glucose-hexokinase trap during the experiment by adding

0.5 ml of the reaction mixture to 4.5 ml 10% perchloric acid to stop the

reaction. Supernatant (2.5 ml) was added to 1 ml 15% KHCO3 solution.

After removal of the precipitated KC104, 0.5 ml of this supernatant was

added to 2.5 ml of the reaction mixture containing 20 units of glucose-6-

phosphate dehydrogenase, 30 pmoles Mg2+, 25 moles TRIS-HC1 buffer (pH

7.8), and 50 moles NADP. The mixture was incubated at room temperature

(25C) for 15 minutes and absorbance was read against a reagent blank at

340 mp in a Beckman DU spectrophotometer modified with a Gilford absorb-

ance indicator.

Determination of enzymic activities

a) NADP-isocitrate dehydrogenase.

To 2.8 ml of reaction mixture containing 2.5 moles NADP, 12 moles

isocitrate, 1.8 pmoles Mn2+, and 25 mmoles TRIS-HC1 buffer at pH 7.5, was

added 0.2 ml of tissue extract supernatantt or sonicated, DMSO treated

mitochondria).




15






b) NAD-isocitrate dehydrogenase.

To 2.8 ml of reaction mixture containing 2.5 pmoles NAD, 12

pmolcs isocitrate, 1.8 smoles Mn2+, and 25 m moles TRIS-HC1 buffer at

pH 7.5 was added 0.2 ml of sonicated mitochondria, which has been

diluted 1:1 (v/v) with DMSO.

c) Isocitrate lyase.

Measurement of isocitrate lyase was attempted by (1) measurement of

the condensation of succinate and glyoxylate by determining the isocitrate

formed with NADP-isocitrate dehydrogenase, (2) measurement of the forward

reaction of glyoxylate reductase, (3) measurement of the forward reaction

by colorimetrically determining the glyoxylate formed.

d) Citrate synthase.

Measurement of this enzyme was by the method described by Bogin and

Wallace (1969).

Measurement of ATP and ADP concentrations

Concentration of ATP was measured by a modified firefly luciferin-

luciferase system employing a Beckman liquid scintillation spectrometer

(Buslig and Attaway, 1969). Each batch of firefly extract was standard-

ized with known amounts of ATP. Concentration of ADP was measured as ATP

after addition of creatine kinase (10 units) and creatine phosphate (10

mg) to 1 ml of the extract, followed by incubation at room temperature for

30 min. After the determination of the total ATP in this preparation the

concentration of ADP was calculated as ADP = ATP total ATP. In every

case determination of ADP was followed by ATP to obtain the results.

Measurement of whole fruit respiration

Carbon dioxide of whole fruit and leaves was measured by a Beckman

infrared C02 analyzer. The fruit or leaves were enclosed in containers




16


equipped with continuous air flow, maintaining approximately 3 inches of

head pressure. The analyzer was connected to the exit port of a series

of solenoids which permitted sequential monitoring of 12 containers (20

minutes each). At the end of the 20 minute period a multipoint recorder

indicated on a chart recorder the C02 concentration of the vessel

measured.

Acidity measurements

Titratable acidity was determined by volumetric measurement, employ-

ing standardized KOH solutions.

Incorporation of 14C02 into whole peeled citrus fruit

The fruits to be tested were carefully peeled and placed in beakers.

The beakers were placed in a 4 liter container that could be tightly

sealed. For 14C02 generation 0.8 mg of barium carbonate (14C) (52.2 PC/

pmole) was also placed in the chamber and 5 ml 2M H2S04 was added through

a port after sealing. The fruit was allowed to remain in this atmosphere

in the dark for 4 hrs. At the termination of the exposure to 14C02 the

jar was flushed for 5 min with compressed air, the exiting gases scrubbed

with 5M KOH to trap all remaining 14C02. Juice was expressed from the

pulp by hand, followed by counting of the radioactivity of a small

portion of the juice.

An aliquot of the juice was fractionated into neutral, anionic and

cationic fractions by chromatography on Dowex-50(H+) and Dowex-l(OH-)

columns. The effluents were analyzed for radioactivity by scintillation

counting.

Radioactivity measurements

Radioactive 14C02, evolved during tracer experiments with Warburg

manometry, was collected in the center well by absorbing into 6M KOH




17





solution adsorbed onto fluted filter paper. Counting was done in 15 ml

toluene containing 0.475% PPO and 0.025% dimethyl-POPOP (Attaway and

Buslig, 1968) by dropping the piece of filter paper in the solution and

using an ambient Beckman liquid scintillation spectrometer.

Aqueous solutions were counted by adding 0.5 ml BBS-2 solubilizer

(Beckman Instruments) to the counting solution.















RESULTS


Titratable acidity and the respiratory activity of various citrus

fruits

Two distinct patterns of organic acid accumulation were observed

during the growth of the citrus fruits studied. These patterns are

represented by sour lemon, sour orange, and bittersweet orange, and are

shown in Fig. 2. The essential difference, besides the levels of organic

acids accumulated, are in the general characteristics of these curves.

Lemon, which accumulates the highest amount of acid, follows a continu-

ously increasing line throughout the growth of the fruit. The curve is

steepest at the initial stages of the growth cycle, slows down about 1/3

full size and continues at a linear rate for the rest of the period. Sour

oranges show an even higher initial rate of accumulation, reaching a maxi-

mum at approximately 1/3 full size, tend to stay at a plateau for a short

period, followed by a slow decline as the fruit increases in size.

Bittersweet oranges accumulate very low concentrations of organic acids,

with an extended plateau, similar to sour orange, declining toward the

end of the growth of the fruit. In all three cases the predominant

organic acid is citric acid.

Respiration of the whole fruits show no particular differences with

the exception of the apparent initial rate at the beginning of the growth

phase. The initial respiratory rate of the sweeter variety shows a

higher level of activity than the sour fruits. Figure 3 shows the


























Fig. 2. Patterns of titratable acid accumulation in bittersweet and sour oranges and in sour lemon.

Arrows indicate mature fruit stage.













6
o Bittersweet orange

5 Sour orange a
Baboon lemon
S4

3
0
<





100 200 300
grams fresh weight

























Fig. 2. Patterns of titratable acid accumulation in bittersweet and sour oranges and in sour lemon.

Arrows indicate mature fruit stage.








o Bittersweet orange
Sour orange
SBaboon lemon


75






50


E


25 .

100 200 300
gramsfresh weight











respiratory activity of the three types examined during their respective

growth cycles. The general pattern shows a continuous decline until

maturity. Examination of mature fruits of various acidity levels indi-

cate no drastic differences in their respiration. Table 2 shows a

comparison of titratable acidity and rate of respiration between the

fruits examined.

Respiratory activity of isolated fruit mitochondria

Mitochondria isolated from various types of citrus fruits at

maturity showed a considerable variation in their ability to metabolize

a series of substrates. Their response to exogenous ADP also showed a

wide range of variation. Generally, it was concluded that the activity

of sour fruits was inferior to that of the sweet fruits. The lower

respiratory control ratio (RCR), the ratio of respiratory activity in

the presence of exogenous ADP vs basal respiratory activity in the

mitochondria, is also an indication of such a difference. The results

obtained from mature fruit are shown in Table 3.

During fruit development respiratory activity of sour and bitter-

sweet orange mitochondria was followed. The results indicate a gen-

erally lower level of respiration of the sour variety with all substrates

tested. Response to added ADP, the RCR, was consistently lower with

mitochondria isolated from the sour fruits at any point of the growth

period. Figures 4 and 5 indicate respiratory activity of bittersweet

and sour orange mitochondria, respectively. Figure 6 shows the response

endogenous respiration to ADP. The most notable feature of the respira-

tory activity of bittersweet orange mitochondria during the period of

observation was the apparent synchrony of activity with every substrate

employed. In contrast the sour orange mitochondria exhibited a biphasic












Table 2. Acidity and respiration of mature citrus fruits
% Titratable Respiration
Type acid/pulp mg C02/kg/hr

Bittersweet orange 0.57 38.50

Sour orange 2.78 25.16

Sampson tangelo 1.95 38.61

Wekiwa tangelo 0.53 41.09

Succary acidless orange 0.09 38.23

Orange de Nice 1.12 34.37

Sweet lemon 0.11 27.91

Baboon lemon .5.24 39.45











Table 3. Respiration of mature citrus fruit mitochondria*

RCR 02 uptake P/0 RCR 02 uptake P/0 RCR 02 uptake P/0

Substrate Bittersweet orange Sour orange Wekiwa tangelo
Succinate 1.37 4.48 1.58 1.00 1.28 0.11 1.14 4.05 0.78
Citrate 2.26 1.58 2.09 0.54 -0.12 5.12 2.04 1.24
a-Ketoglutarate 2.20 4.74 2.40 3.00 0.60 0.47 1.53 2.85 1.74
Pyruvate+malate 1.83 4.32 2.68 0.11 0.08 0 2.14 2.89 1.11


Sampson tangelo Baboon lemon Sweet lemon
Succinate 1.53 2.81 0.26 0.26 0.09 0 1.42 3.90 1.11
Citrate 5.82 1.22 0.34 0.26 0.08 1.77 1.38 1.87
aI-Ketoglutarate 1.23 2.40 0.87 1.87 2.67 2.28
Pyruvate+malate 1.75 2.58 0.43 0.01 0 0 0.90 1.59 3.32


Valencia orange Acidless orange Marsh grapefruit
Succinate 7.20 1.92 2.17 3.16 1.48 1.32 2.77 1.29
Citrate 3.94 4.00 1.40 0.66 1.08 9.33 0.74 2.12
a-Ketoglutarate 6.62 3.00 1.55 2.87 2.19 2.18 1.73 2.58
Pyruvate+malate 3.80 3.88 1.71 1.54 2.07 1.26 1.40 2.49

02 uptake is patoms/hr/mg mitochondrial protein.





























Fig. 4. Respiration of bittersweet orange mitochondria.

















endogenous

o citrate
succinate

2 o -ketoglutarate

/ pyruvate + malate


100 200
grams fresh weight


300


h--
c

o




0)
i5






E

E



L
t-





























Fig. 5. Respiration of sour orange mitochondria.













. * endogenous
0 : citrate
c 5 / succinate
a. / \ o o-ketoglutorate
/ \ pyruvate + malote


g I \
/ / t

0 / K /".-\ /"
0 .... .40 I
100 200 300
grams fresh weight



























Fig. 6. Respiratory control ratio changes during development.














x Bittersweet orange
o Sour orange











x x




o





100 200 300
grams fresh weight


0

2
0

0
0



0
Ol











type of activity with all substrates showing synchrony at the beginning

of the growth curve, succinate and pyruvate + malate showing a second

smaller increase in activity while the rest of the curves hit minima.

The RCR (Fig. 6) curves of endogenous respiration in both instances

show similar shapes, but the bittersweet orange mitochondria indicate

a considerably higher respiratory control by ADP at all comparable

points.

Enzymic activities of fruit pulp during growth

For evaluation of some of the enzymes contributing to the syn-

thesis and to the degradation of citric acid, which is the predominant

acid in these fruits, the enzymes citrate synthetase, 2 isocitrate de-

hydrogenases, NADP and NAD specific, and isocitrate lyase were selected.

Citrate synthetase activity is shown in Figures 7 and 8 for bitter-

sweet and sour oranges, respectively. The curves indicate a comparable

initial activity in both fruit types, with bittersweet orange having a

slightly higher initial activity. As the fruits grew, both fruits

showed an increase in the enzymic activity, followed by a decline and

an apparent plateau for the bittersweet orange. The sour orange shows

a slight decrease in activity, followed by another increase in activity.

Total enzyme per fruit increases with both varieties, however the final

level was considerably higher in the sour fruit.

Isocitrate dehydrogenase (NADP-specific) showed a somewhat differ-

ent pattern. In the sour fruit, there is an initial rapid decline,

reappearance and another decline, ending in a constant level with fruit

from about 2/3 full size to maturity. The bittersweet orange shows an

initial plateau, followed by a rapid decline at approximately 1/2 full

size, ending in a slow decrease toward maturity. Figures 9 and 10 show






























































Fig. 7. Citrate synthetase activity of bittersweet oranges.


__






























































200
groms fresh weight


c
E






x )


-100.-








5E
o

5 O:,


300









































Fig. 8. Citrate synthetase activity of sour oranges.





36


x
/
/
/
,,/ ,'.,....""" x


100 200
grams fresh weight


300








































Fig. 9. NADP-isocitrate dehydrogenase activity of bittersweet oranges.



















0.2- -4







-3







. 2 E

.. .. .
E \













100 200
grams fresh weigh t




























Fig. 10. NADP-isocitrate dehydrogenase activity of sour oranges.












0.15r -300











0 0



o 0
0.10- -200:


















100 200 300
grams fresh weight
0.05- -100 o









100 200 300
grams fresh weight




41




rates of activity and total activity per fruit. The total activity

curves are very similar in both fruit types, although bittersweet

seems to have higher activity over all.

Isocitrate dehydrogenase (NAD-specific) activity seems to be

quite similar in both types of fruit. The levels of activity are

comparable. Figures 11 and 12 show rate and total activity.

Isocitrate lyase was not found in any of the citrus fruits

examined thus far.

Concentrations of ATP and ADP in fruit pulp

The concentration of ATP and ADP was measured in the three types

of fruit with the organic acid accumulative patterns described earlier.

The results are shown in Table 4. The most notable information that

can be obtained from these figures is the initially higher level of

both nucleotides in the pulp of the sour lemon fruit, lower in the sour

orange, and finally lowest in the bittersweet orange. At the end of

the growth period, the order is reversed, with bittersweet showing the

highest observed concentration of both.

Metabolism of 14C-citrate and 14C-pyruvate by isolated mitochondria

and whole juice sacs

In these experiments the mitochondria were isolated from mature

citrus fruits. The results are shown in Table 5. It may be noted that

as before respiratory efficiency of the bittersweet orange mitochondria

is greater than that of the sour orange. These results are in agreement

with earlier observations shown in Figures 4 and 5, for comparable

stages of fruit growth.

Table 6 lists the results obtained with whole, isolated juice

sacs. These results once again reconfirm the higher efficiency of the

less acid fruit to metabolize Krebs cycle intermediates.




























Fig. 11. NAD-isocitrate dehydrogenase activity of bittersweet oranges.












15 600






.-






E 5 .200







100 200 300
grams fresh weight
grams fresh weight








































Fig. 12. NAD-isocitrate dehydrogenase activity of sour oranges.


















11000
I A


800


600

:t



"5




o




x


45


cn





0

E
03
S











Table 4. Concentration of ATP and ADP during development in Baboon lemon, bittersweet and
sour oranges
Average weight ATP ADP ATP
per fruit (g) m I moles/g pulp m nmoles/g pulp ADP

Baboon lemon
17.95 143.6 180.5 0.78
147.00 48.9 35.2 1.39
189.25 53.6 35.9 1.49
266.00 38.4 44.3 0.87
357.05 37.6 31.8 1.18
Bittersweet orange
35.05 62.8 130.9 0.48
52.17 48.0 56.1 0.86
96.00 56.2 32.5 1.73
120.10 37.0 33.2 1.12
176.25 38.9 39.9 0.98
Sour orange
18.40 121.2 113.1 1.07
37.60 110.8 44.2 2.50
55.00 64.6 60.7 1.06
137.00 40.0 54.2 0.74
224.00 30.3 16.1 1.88












Table 5. Metabolism of radioactive citrate and pyruvate by isolated mitochondria
Sampson tangelo Wekiwa tangelo
Substrate 02 uptake 14C02 released 02 uptake 14C02 released
atoms* cpm* atoms" cpm*

Citrate-14C(1,5)
-ADP 0.17 33 0.40 34

Citrate-14C(1,5)
+ADP 1.22 127 2.04 233

Pyruvate-14C (2)
+malate-ADP 1.47 26 1.35 27

Pyruvate-14C(2)
+malate+ADP 2.58 30 2.89 34

Both are per hr/mg mitochondrial protein














Table 6. Metabolic activity of whole juice sacs
Bittersweet orange Sour orange
Substrate 02 uptake 14C02 released 02 uptake 14C02 released
atoms" cpmu atoms* cpm*

Endogenous 1.83 1.65

Citrate-14C 2.03 2.84 1.47 2.73

Pyruvate-14C 2.82 0.64 1.49 0.61

Both are per hr/g juice vesicles







49


Dark fixation of 14C02 by whole peeled fruit

The fixation of 14C02 by peeled bittersweet and sour oranges was

examined. The results indicate a lower extent of incorporation of radio-

activity into the juice of sour oranges. In Table 7 the level of incor-

poration is indicated, with the results obtained on fractionation of the

extracted juice into neutral, cationic and anionic fractions. It is

assumed that these fractions represent largely sugars, amino acids and

organic acids, respectively. The sour orange seems to be able to incor-

porate a greater proportion of dark fixed carbon dioxide into the neutral

fraction, and a lower proportion into the amino acid fraction than

bittersweet. Incorporation into organic acids seems to be to approxi-

mately the same level, although due to the differential in acidity levels

between the two, a greater percentage of the acids in bittersweet oranges

are labeled.

The effect of arsenate on mitochondrial activity of Valencia

oranges

For this experiment Valencia oranges were selected instead of the

commercially sprayed grapefruit, due to the greater sensitivity of these

fruits to the effects of arsenate. In Table 8 the results of these ex-

periments are shown. It is apparent from the results obtained that a

concerted reduction in respiratory associated activity occurs, as both

oxygen uptake and phosphorylation were decreased. The decrease in oxygen

uptake seems to be proportional to the decrease in phosphorylation,

rather than causing uncoupling attributed to arsenate.


















Table 7. Incorporation of 14C02 into whole peeled fruit

Total 14C incorporated/ml juice 14C incorporated cpm/ml
cpm Neutral Cationic Anionic


Bittersweet orange 23815

Sour orange 20552

Results are expressed as incorporation/4 hrs


9535 1570 6625

11856 653 6403













Table 8. Respiration of Valencia orange mitochondria*

02 uptake Pi esterified P/0
patoms/hr pmoles/hr corre

0.41 1.33 3.24
6.61 5.18 0.78
2.38 3.56 1.50
5.59 6.41 1.15
4.05 5.27 1.30


p/QA*


P/0*
cted for endogenous


0.62
1.13
0.98
1.08


Substrate
+ 10-2M As04
Endogenous 0.47 1.34 2.85
Succinate 6.03 3.98 0.66 0.48
Citrate 2.12 2.46 1.16 0.68
a--Ketoglutarate 2.44 3.39 1.39 1.04
Pyruvate + malate 2.46 3.16 1.29 0.91

Results expressed are per mg mitochondrial protein

These results represent activity of mitochondria above basal rate


Subs rate
-As04
Endogenous
Succinate
Citrate
a-Ketoglutarate
Pyruvate + malate














DISCUSSION

The experiments were designed to investigate the problem of citric

acid accumulation in citrus fruits. This phenomenon involves several

facets of fruit metabolism, some of which are incidental and some are

integral parts of the organic acid accumulation process.

The early work on organic acid accumulation in citrus (Bartholomew,

1923) was concerned mainly with the patterns of increase of titratable

acids. Later workers (Sekhara Varma and Ramakrishnan, 1956; Sinclair and

Ramsey, 1944; Harding and Fisher, 1945; Bain, 1958; Ting and Deszyck,

1959; Clements, 1964 a, b; Rasmussen, 1964; Ting and Vines, 1966) examined

the quantitative distribution of some of the organic acids found in the

fruits. From such investigations it became apparent that citric acid was,

in most instances, the predominant organic acid in citrus (Erickson, 1968).

Biosynthetic pathways leading to the formation of citric acid are well

known since the pioneering work of Krebs and Johnson (1937), who demon-

strated this acid as the integral part of aerobic respiratory pathways in

tissue homogenates. Now it seems reasonable to conclude that all living

organisms form this compound to varying extents.

Since organic acid metabolism and respiration are very closely

connected phenomena, the sequence of experiments is purported to re-

flect a series of observations, starting on the outside of the fruit with

gross respiratory patterns, along with measurement of titratable acidity

during growth (Figures 2 and 3, Table 2). Metabolic activity that can be

concluded from these results indicates the reduction of respiration as


52




53


the fruit grows, along with the reduction of surface/volume ratio, and

in all cases, the increase of acidity. Since the results of respiration

do not parallel the accumulation pattern, further examination on the

subcellular level, such as isolated mitochondrial respiration, gives a

considerably more accurate indication of respiratory activity as linked

to acidity. Considering the various citrus fruits (Table 3), each with

different acidity characteristics, it may be safely concluded that a

correlation exists between acidity level and the respiratory activity

of isolated mitochondria. Seasonal variations are reflected in exami-

nation of RCR of bittersweet and sour oranges (Fig. 6) and respiratory

activity of both types of mitochondria, respiring various substrates

(Fig. 4, 5). The apparent synchrony of the metabolism of substrates by

bittersweet orange mitochondria and the asynchrony observed with sour

orange mitochondria, along with the efficiency of metabolism of these

substrates is indicative of some differences in the controlling mechanism

of respiration. It is readily apparent, that citrate is metabolized by

sour varieties at a lower rate than the "normal", not quite so acid,

varieties. In fact respiration of sour orange mitochondria show a tend-

ency to be inhibited by the substrate concentration used below the

endogenous level in the presence of citrate. It is also interesting to

note, that succinate and pyruvate + malate (Fig. 5), which all precede

citrate in the normal sequence of respiration, are the efficient sub-

strates at this stage of growth while citrate, and subsequently t-

ketoglutarate, are poorly metabolized at the same time. This could re-

sult in the accumulation of citrate.

Confirmation of the higher efficiency of less acid varieties was

obtained by metabolizing 1C-pyruvate, and 1C-citrate both by isolated










mitochondria alone, and also by whole juice sacs (Tables 5, 6). In

these experiments the juice sacs showed unexpectedly higher rates of

equilibration between exogenous substrate and the cell endogenous re-

serves with sour orange, while both substrates caused some depression

of the respiratory rate. With juice sacs from bittersweet orange, this

equilibration was considerably slower, resulting in an 02/CO2 ratio of

0.36 with citrate and 2.20 with pyruvate versus 0.27 and 1.22 respect-

ively for sour orange.

The higher efficiency of metabolism of a less acid variety is

especially significant when further results are examined (Table 5).

These two tangelos are very closely related. Sampson was the maternal

parent of Wekiwa, but they are quite distinct as far as their acidity

levels (Table 1). It is very convincingly shown that the respiratory

activity of these mitochondria is very efficiently controlled in Wekiwa

by exogenous ADP, but not nearly as well in Sampson. With citrate as

substrate, Wekiwa mitochondria respire and degrade citrate about twice

as fast as Sampson mitochondria, while the absolute level of pyruvate

metabolism changes very little, indicating some possible control of

pyruvate entry into the mitochondria when applied exogenously. This

result is not very clearly explained at this time.

The enzymic reactions involved in the biosynthetic and degradative

pathways of citrate were the first objects of investigation in citrus

fruits. Initial observation by Sekhara Varma and Ramakrishnan (1956)

suggested that citrus fruits are capable of synthesizing the organic

acids found in them. Erickson (1957) came to a similar conclusion

through grafting sour lemon on sweet lemon stock and the reverse, with-

out significantly altering organic acid composition of the fruit. A





55


series of articles from California (Huffaker and Wallace, 1959; Clark

and Wallace, 1963; Bogin and Erickson, 1965; Bogin and Wallace, 1966 b)

indicated the involvement of metabolic blockage by an inhibitor, which

seemed to form at a side reaction, along with the reversal of some of

the Krebs cycle reactions, as the cause of the accumulation of citric

acid. The reactions that were involved, individually at least, are

capable of the synthesis of citric acid or an immediate precursor.

Enzyme activities involved in the synthesis and degradation of

citric acid are shown with increasing size (maturity) of bittersweet

and sour orange fruits (Figs. 7 to 12).

Citrate synthetase activity of bittersweet orange (Fig. 7) seems

to be quite high at the beginning stages of growth, reaches a climax

at about 1/4 maximum size, declines slowly to a plateau about 1.3-1.4

pmoles/min/g pulp. Total activity present in the pulp increases nearly

linearly until maturity. Sour oranges present an entirely different

citrate synthetase activity pattern (Fig. 8). The initial activity is

lower than in the bittersweet orange, but it increases rapidly, slows

somewhat at 1/2 full fruit size, and again shows a very rapid increase.

It seems to reach a plateau perhaps at maturity, considerably higher

than bittersweet orange. Total activity increases rapidly after an

initial lag, slowing at maturity, so as to present a sigmoid-shaped

curve. The differencein activity during growth is a good indication of

a higher metabolic control of this enzyme during development.

The two enzymes intimately involved in the degradative pathway of

citrate follow aconitase, which establishes a ready equilibrium between

citrate, cis-aconitate and isocitrate. Isocitrate degradation is

readily accomplished by either NADP or NAD specific isocitrate






56


dehydrogenases. Both are present in citrus (Buslig and Attaway, 1968 a;

Buslig, unpublished). Activity of the NADP-specific isocitrate dehydro-

genase in bittersweet orange shows a higher initial activity which

slowly declines until the mid-point of growth, drops very rapidly to 1/3

initial level and declines further slowly until maturity. Sour orange

shows a rapid drop initially, followed by a transient rise, falling back

to a level about equivalent to the first low point. Total activity in

both instances seems to follow the same general pattern, although higher

activity can be observed with bittersweet throughout the growth cycle

(Figs. 9, 10).

NAD-isocitrate dehydrogenase activities are quite similar in both

fruits, differing towards maturity as sour orange NAD-isocitrate dehydro-

genase declined while bittersweet orange actually showed an increase

(Figs. 11 and 12).

It is interesting to observe that bittersweet orange showed in-

creasing activity of the degradative isocitrate dehydrogenases after

citrate synthetase reached its peak, while sour oranges show an initially

higher activity of these enzymes, but both decline as citrate synthetase

continues to increase.

These results indicate that the activity of citrate synthetase

alone may be sufficient to account for the rise in acidity in sour

oranges, while the closely related bittersweet variety seems to have

control of not only the general respiratory pattern during growth but

citrate synthetase as well.

Mitochondrial asynchrony of metabolism was observed in sour orange,

which along with the enzyme activity patterns could account for the ob-

served accumulation pattern of citric acid. These results are in






57


agreement with previous investigators (Erickson, 1958) who observed

similar patterns of accumulation. However, the present information on

enzymic patterns would seem to indicate that at least part of the

accumulation of citrate could be due to activities of citrate synthetase

and isocitrate dehydrogenase levels without a complex inhibitory system

as they described.

Huffaker and Wallace (1959) and Clark and Wallace (1963) indicated

in their papers that reversal of some decarboxylation reactions may

account for accumulation of citrate. Dark fixation of 14C02 by the two

types of oranges (Table 7) is in agreement with their results; however

it is obvious that such levels of incorporation, especially the close-

ness of the results, will not account for the great differences in acidity

observed.

In earlier work with citrus no attempt was made to explain the

accumulation of citric acid to a level, which if present in the mito-

chondria or cytoplasm without some type of barrier, that would be quite

deleterious to the entire fruit. Recently Abou-Zamzam and Wallace (1970)

examined adenylate kinase, one of the enzymes which is possibly involved

in energy balance within the cell. Such a reaction could shed light on

the problem of accumulation, as it may be able to account for the energy

requirements to remove the high concentration of citric acid from vul-

nerable areas to the vacuole by active transport. Adenosine nucleotides,

especially ATP, were found to be higher during growth in sour orange and

baboon (sour) lemon than in bittersweet orange (Table 4). In lemons a

similar situation exists (Abou-Zamzam, Wallace and Motoyama, 1970). As

organic acids accumulate, ATP levels decline in all cases. ATP/ADP




58


ratios were found quite variable, generally increasing during the great-

est rate of increase in acidity. Hydrolysis of ATP may be able to supply

the needed energy for translocation of the acids into the vacuole, where

it may be accumulated without deleterious effects. Such compartmentation

would require additional energy for maintenance of high H+ concentrations

in the vacuole. Assuming a concentration gradient of 4 (pH within

vacuole 3.5, in cytoplasm 7.5), energy required to establish this gradi-

ent would be (at 20'C):
10-3.5
G = 2.303 RT log 1
10-7.5


G = 2.303 X 1.98 X 293 X 4 = 5344 cal/mole
5344/8000 = 0.67 moles ATP

hydrolized to establish the gradient (Lehninger, 1965). Of course the

higher the accumulation, the higher the requirement.

Compartmentation and active transport would agree very well with

the present results. It is obvious that genetically controlled enzyme

systems operate in citrus, especially on the basis of closely related

varieties. The genetically controlled active transport system (Ranson,

1965) and evidence presented by Oaks and Bidwell (1970) would also be

supported by these findings.

In citrus fruit some control of acidity can be achieved by the

application of post-bloom sprays of lead arsenate (Reitz, 1949). It was

suggested, that the mechanism of action of this compound is that of an

uncoupler, removing oxidative control of phosphorylation in the mitochon-

drial (Vines and Oberbacher, 1965). Repetition of these experiments

indicated a minimal degree of uncoupling (Table 8). The results pointed




59





to competition of arsenate with phosphate, as about an equal degree of

reduction was obtained in both oxygen uptake and phosphorylation.

In conclusion, the present results indicate the control of citric

acid accumulation to lie in part in the control of the enzymes citrate

synthetase and the subsequent degradative dehydrogenases. Adenosine

nucleotides play a controlling influence on the concerted respiratory

mechanism of the Krebs cycle, with a possible role in the transport

mechanism from the mitochondria and, cytoplasm to the vacuole. It does

not seem likely that dark C02 fixation plays a major role in acid

accumulation.















SUMMARY


Respiratory and metabolic interrelationships involving biosynthesis

and degradation of citric acid in citrus fruits were studied. The data

obtained indicate the following:

1. Citrate accumulation in part is due to altered control of

citrate synthetase in the more sour variety.

2. Energetics of acid accumulation may be accounted for by the

higher levels of ATP and higher ATP/ADP ratios in sour fruits during

the greatest increase in citric acid content.

3. Asynchrony in favor of citrate production exists with the en-

zymes examined in the sour fruit, while the sweeter variety shows a

reasonable synchrony.

4. Control of respiration and of Krebs cycle activity by

exogenously applied ADP in the mitochondria in citrus fruits was more

efficient in the sweeter variety.

5. Variations between the sweet and sour varieties seem to be

due to a primary genetic control of the enzymes involved.

6. The action of arsenate on respiration in citrus fruits seems

to be due to competition with phosphate. In contrast earlier interpre-

tation considered it to be an uncoupler of oxidative phosphorylation.


60














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and sour lemon fruits. J. Amer. Soc. Hort. Sci. 95: 199-202.

Abou-Zamzam, A. M., A. Wallace, and E. Motoyama. 1970. Measurement of
the endogenous levels of adenosine nucleotides in sweet and sour
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Attaway, J. A., and B. S. Buslig. 1968. Conversion of linalool to
a-terpineol in citrus. Biochim. Biophys. Acta 164: 609-610.

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Bartholomew, E. T. 1923. Internal decline of lemons. II. Growth rate,
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Beevers, H. 1961. Respiratory Metabolism in Plants. Harper and Row,
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Beevers, H., M. L. Stiller, and V. S. Butt. 1966. Metabolism of the
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Bogin, E., and L. C. Erickson. 1965. Activity of mitochondrial prep-
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Bogin, E., and A. Wallace. 1966a. C02 fixation in preparations from
Tunisian sweet lemon and Eureka lemon fruits. Proc. Amer. Soc.
Hort. Sci. 88: 298-307.

Bogin, E., and A. Wallace. 1966b. Organic acid synthesis and accumu-
lation in sweet and sour lemon fruits. Proc. Amer. Soc. Hort.
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BIOGRAPHICAL SKETCH


Bdla Stephen Buslig was born on August 27, 1938 in Budapest, Hungary.

He attended primary and secondary schools there. In 1956, following the

Revolution,he escaped to Austria. Subsequently he lived in England and

Canada, where he completed his undergraduate training at Queen's University

at Kingston, Ontario, receiving a degree of Bachelor of Arts (General) in

1962 with concentration in biology and chemistry. Following graduation he

attended Western Reserve University as a N.I.H. research fellow in micro-

biology. In 1963 he enrolled in the Florida State University Graduate

School pursuing a program of study in genetics. In 1967 he obtained the

degree of Master of Science in biology, with emphasis on the molecular

genetics of bacteria. Soon after graduation he started working as a

chemist for the Florida Citrus Commission at the Lake Alfred Citrus Experi-

ment Station. At the same time he proceeded with further studies in the

Department of Fruit Crops at the University of Florida, to obtain the

degree of Doctor of Philosophy, concentrating on fruit physiology.

He is a member of the Canadian Society of Plant Physiologists,

American Chemical Society, Genetics Society of America, American Associ-

ation for the Advancement of Science, New York Academy of Sciences,

Florida State Horticultural Society and the American Society for Horti-

cultural Science.

He is married to the former Bertha Horsfall and has two children.

Presently he is employed by the Scientific Research Division of the

Florida Department of Citrus as a Research Biochemist.











I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Robert H. Biggs, Chair an
Professor (Biochemist), Fruit Crops


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Jbhn A. Attaway, Co-chairman /
Professor (Chemist), Citrus Experiment Station


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Alfred H.
Professor (HoT cu-turist), Fruit Crops


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




William F. Newhall
Professor (Biochemist), Citrus Experiment Station










I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Davi S. Anthony
Professor (Biochemistry), Botan



This dissertation was submitted to the Dean of the College of Agriculture
and to the Graduate Council, and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.

December, 1970





Dean, College of Agricul





Dean, Graduate School




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