ASSIMILATE PARTITIONING AND ENZYMES OF ORGANIC ACID
METABOLISM IN FRUIT OF CALAMONDIN AND LOW-ACID GRAPEFRUIT

BY

CHUNG-RUEY YEN

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

UNIVERSITY OF FLORIDA

ACKNOWLEDGEMENTS

I wish to express my deepest appreciation to Dr. Karen E. Koch, my
advisor, for her thoughtful guidance, friendship, and patience
throughout the research. I wish to extend my sincere appreciation to
the committee members, Dr. Wayne Sherman, for his encouragement; Dr.

C. Jack Hearn, for his kind supply of plant materials; Dr. Frederick
Davies, for his help and friendship and as a substitute committee
member during Dr. Sherman's sabbatical leave in Australia; Dr. Thomas
Humphreys, for his discussion in my research; and Dr. Kenneth Boote,
for his good instruction in plant physiology which provide some basic
groundwork for my research.

I am very grateful to Wayne Avigne for technical assistance
throughout the research, also special thanks are offered to Cadance
Lowell and Jonathan Crane, Dave Douds and Pat Tomlinson, for good
suggestions and discussion in my research.

I wish to thank the financial support of the Council of
Agriculture, Republic of China. I would like to thank the support and
suggestion of Mr. Liang Li, Director of Chia-Yi Agricultural
Experiment Station, Dr. Shue-cheng Lin, Chairman of Department of
Horticulture, Taiwan Agricultural Experiment Station and Dr.

Ching-lung Lee, Chief, Horticultural Division, Council of Agriculture.

Finally, I would like to express my deepest thanks to my wife,
Li-lin, for her help and patience in every aspect during the time in

ii

the United
whenever I

States, and my parents, who raised me and gave support,
need it.

iii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT ii

LIST OF TABLES vi

LIST OF FIGURES vii

ABSTRACT ix

CHAPTER

I INTRODUCTION 1

II LITERATURE REVIEW 5

Assimilate Partitioning in Citrus 5

Organic Acids in Citrus Fruits 8

Organic Biosynthesis in Citrus Fruits.... 10

Arsenate Treatment in Citrus.... 13

Enzymes Associated with Organic Acid Metabolism...... 15

III DISTRIBUTION OF ASSIMILATES PRODUCED DURING LIGHT

AND DARK 1 XO FIXATION BY DEVELOPING GRAPEFRUIT
AND ADJACENT SOURCE LEAVES 21

Introduction.... 21

Materials and Methods 23

Results and Discussion 25

Conclusions 44

IV ORGANIC ACIDS FROM LIGHT AND DARK CO FIXATION BY

LEAVES AND FRUIT OF LOW-ACID CITRUS MUTANTS 45

Introduction 45

Materials and Methods. 47

Results 49

Discussion. 59

Conclusions..... 62

V EFFECTS OF ARSENATE ON ACIDITY AND LONG-TERM

ASSIMILATE PARTITIONING IN CALAMONDIN 64

Introduction 64

Materials and Methods... 66

Results and Discussion 68

iv

Conclusions

76

VI ENZYMES OF ORGANIC ACID SYNTHESIS AND METABOLISM

DURING DEVELOPMENT OF NORMAL AND LOW ACID GRAPEFRUIT
MUTANTS 78

Introduction 78

Materials and Methods 81

Results 83

Discussion 98

Conclusions 104

VII CONCLUSIONS 105

LITERATURE CITED 108

BIOGRAPHICAL SKETCH 119

v

LIST OF TABLES

Table

Page

3.1

14

Contribution of C-assimilates derived from various

sources to developing grapefruit

3.2

Parameters utilized for assessmenrts of relative C
contributions by various sources to growth of grapefruit

. 30

3.3

The relative overall C contribution by various sources
to growth of grapefruit

3.4

Distribution of ethanol-soluble 1^-photosynthates in
fruit tissues 1 h and 24 h after CC^ exposure

4.1

The ratio of citric to malic acid in juice tissues
during development of fruit on normal and low acid
grapefruit clones

. 53

4.2

The contribution of various sources to organic acids
in juice tissues of normal and low acid grapefruit
during fruit development.....

5. 1

Arsenate treatments and acidity (meq g ^ fresh wt) of
calamondin fruit

5.2

Total dpm recove^d from 3-month old calamondin fruit
after different CO^ treatments

5.3

14

C-assimilate distribution among tissues of arsenated
and non-arsenated calamondin trees

5.4

14

C-assimilate partitioning into organic acid fraction
of juice tissues of arsenated and non-arsenated
calamondin fruit

. 75

6. 1

The rate of organic acid accumulation and ratio of
citrate synthase to aconitase in extracts from juice
tissues of normal- and low-acid grapefruit during
development

LIST OF FIGURES

Figure

3.1

3.2

3.3

3.4

4.1

4.2

4.3

6.1

6.2

6. 3

6.4

6.5

Page

Developmental changes in fresh and dry weight of

tissues in seedless grapefruit clone 27

Developmental changes in partitioning and distribution
of C-assirailates in seedless grapefruit mutant 34

Partitioning and distribution of fruit
14C-photosynthate^in grapefruit mutant between
and 7 days after CO^ treatments

Distribution of dark fruit

14

^apefruit mutant between 1,

CO^ treatments.

C-assimilates in
6, and 24 h after

1

The acid concentration and total titratable acidity
in juice tissues of developing normal and low acid
grapefruit mutants.....

14

Developmental changes of percentage of C-assirailate
partitioning into organic acid fraction in juice
tissues

40

43

51

55

The amount of organic ^id synthesis derived from

various sources after fixation 58

Acid levels during development of flavedo, albedo,
and juice tissues of seedless normal and low acid
grapefruit mutants 85

Protein cotents during development of flavedo, albedo,
and juice tissues of seedless normal and low acid
grapefruit mutants 88

Phosphoenolpyruvate carboxylase during development

of flavedo, albedo, and juice tissues of seedless

normal and low acid grapefruit mutants 90

Malate dehydrogenase during development of flavedo,
albedo, and juice tissues of seedless normal and low

acid grapefruit mutants 93

Citrate synthase during development of flavedo, albedo,
and juice tissues of seedless normal and low acid
grapefruit mutants 95

vii

Aconitase during development of flavedo, albedo, and
juice tissues of seedless normal and low acid
grapefruit mutants

viii

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

ASSIMILATE PARTITIONING AND ENZYMES OF ORGANIC ACID
METABOLISM IN FRUIT OF CALAMONDIN AND LOW-ACID GRAPEFRUIT

By

Chung-Ruey Yen
December, 1987

Chairman: Dr. Karen E. Koch

Major Department: Horticultural Science (Fruit Crops)

Organic acid formation in citrus fruit was characterized with

14

respect to light and dark CO^ fixation and enzymes of organic acid

metabolism to quantify the sources of organic acids and identify

possible mechanisms controlling acid accumulation in citrus fruit.

Results were compared throughout development of grapefruit from

normal- and low-acid clones and also in fruit of calamondin trees

treated to reduce fruit acidity.

14

The C-assimilates translocated from leaves to fruit were

deposited primarily in juice tissues regardless of whether ^CC^

fixation had occurred in light or dark. The majority of
14

C-photosynthates originating in fruit remained in peel tissues.
During most of development, assimilates from dark fixation by

fruit were localized primarily in juice tissues and little subsequent
redistribution occurred. Leaf photosynthates were the primary C source

ix

for fruit growth, while the fruit photosynthates mainly contributed to
growth of peel (flavedo and albedo).

Sources of organic acids were the same as those of total C only in
very young fruit. Leaf photosynthates were the primary source of
organic acids throughout early fruit development. However, with fruit
expansion, assimilates from dark CC^ fixation by fruit became one of
the major, and possibly the predominant, source of organic acids. This
was evident despite the minimal contribution to fruit C by dark C02
fixation.

Assimilate partitioning into organic acids was altered in leaves,

but not fruits of arsenate-treated calamondin trees, and a greater
14

portion of leaf C-photosynthates was recovered in fruits. The
contribution of fruit photosynthesis to growth of calamondin juice
tissues appears to be greater than that of grapefruit (23% vs. less
than 4%) and may be related to differences in peel thickness.

Examination of enzyme activities indicated that dark CO,, fixation
via the combined reactions of phosphoenolpyruvate carboxylase
(PEPc) and raalate dehydrogenase (MDH) was an important source of
organic acid synthesis, but overall control of organic acid
accumulation apparently occurred at steps other than the PEPc-MDH
pathway. The greater activity of citrate synthase and lesser activity
of aconitase in tissues with high acid levels, as well as the reverse,
indicated that these two reactions together may be important in the
control of synthesis and degradation of citrate in citrus fruit.

x

CHAPTER I
INTRODUCTION

Organic acids play a central role in plant metabolism. Citric acid
and malic acid, which are the main organic acids in citrus fruit, are
not simply the intermediates of respiration. Malic acid is also
involved in photosynthesis (Edwards and Huber, 1981; Ting, 1985), pH
balance (Davies, 1979; Bown, 1985), and nitrogen assimilation (Raven
and Smith, 1976; Robinson, 1983; Lance and Rustin, 1984). Many fruits
are particularly rich in organic acids which are usually dissolved in
the cell water, either as free acids or combined as salts, esters or
glycosides (Ulrich, 1970).

Acid content is an important quality factor in fruit and can be
affected by genetic, developmental and environmental factors. The
ratio of soluble solids to total acids is also a major criterion of
maturity for citrus and certain other fruits (Ulrich, 1970; Sinclair,
1984). Citric and malic acids predominate in citrus fruit, but oxalic,
malonic, succinic and other minor acids can also be found (Kefford and
Chandler, 1970; Monselise and Galily, 1979). Citric acid is
predominant in fruit of sour citrus species while malic acid is more
prevalent in those with sweeter fruit (Kefford and Chandler, 1970).
Organic acids accumulate throughout the development of citrus fruit
(Ting and Vines, 1966; Erickson, 1968; Kefford and Chandler, 1970;
Sinclair, 1984), but the rate and timing of accumulation vary with
different species.

1

2

Three hypotheses have been proposed to account for the origin of
organic acid accumulated in fruit:

1. Organic acids are formed within fruit via metabolism of
photosynthates transported from leaves (Varma and Ramakrishnan, 1956;
Ramakrishnan, 1971).

2. Organic acids might be transported directly from leaves or roots as
in grapevine (Ulrich, 1970).

3. Organic acids are products of fixation by fruit, either
directly from phosphoenolpyruvate(PEP ) carboxylase activity in juice
tissues (Huf faker and Wallace, 1959), and/or metabolism of
carbohydrates produced during photosynthesis in fruit.

Recent research tends to favor the first hypothesis. Evidence
indicates that some organic acids are produced from photosynthates
imported into fruit and accumulation can presumably occur if there is
an block in metabolism (Buslig, 1971; Ramakrishnan, 1971; Kubota and
Akao, 1973a). Overproduction of organic acids and/or slow utilization
would need to be followed by uptake and storage inside vacuoles.

Still, no quantitative data describe the extent to which this occurs
or confirm a specific source of fruit acids.

Less data exist on transport of organic acids from leaves to
fruits. Organic acid levels are quantitatively a minor constituent of
sieve-tube sap and initially seem insufficient to explain the large
accumulation in fruit (Ziegler, 1975). However, organic acids comprise
only 1.2% to 1.8% of the fresh weight in grapefruit and 4.4% to 6.4%
in lemon juice (Sinclair, 1984). They may make up 2.0 to 3.2 mg/ml of
the phloem sap (Hall and Baker, 1972). A major portion of grape
acidity has been found to originate in this way (as reviewed by

3

Ulrich, 1970).

Fixation of CO^ in fruit can also give rise to organic acids,

especially if initial carboxylation occurs via PEP carboxylase

(Huffaker and Wallace, 1959; Bean and Todd, 1960). This enzyme is

known to be active in citrus fruit (Huffaker and Wallace, 1959; Bogin

and Wallace, 1966a). Huffaker and Wallace (1959) first suggested that

dark C02 fixation could be related to acid accumulation in citrus

fruit and that PEP carboxylase was a key enzyme in C02 fixation. Bean

and Todd (1960) further identified labeled organic acids in young

14

lemon fruit after dark C02 fixation. However, the relationship
between amounts of CO^ fixation and fruit acidity differed depending
on fruit tissues examined and methods used (Clark and Wallace, 1963;
Bogin and Wallace, 1966a). Still, overall photosynthetic rates (via
RuBP carboxylase + PEP carboxylase) were greatest in young citrus
fruit during the time when much organic acid accumulation occurred
(Kidd and West, 1947; Bean and Todd, 1960; Todd et al. , 1961;
Kriederaann, 1968a; Pandey and Farmahan, 1977; Moreshet and Green,

1980; Chauhan and Pandey, 1984). Total fruit photosynthesis is
believed to contribute little to development of the organ (Kidd and
West, 1947 ; Todd et^ al . , 1961, Schaedle, 1975; Chauhan and Pandey,
1984), but the amount of organic acids derived either directly or
indirectly from this process within fruit is unknown.

Photosynthate partitioning in whole plants has been extensively
studied (as reviewed by Wardlaw, 1968; Wareing and Patrick, 1975;
Geiger and Giaquinta, 1982; Daie, 1985; Delrot and Bonneraain, 1985),
but little attention has been directed toward the identity, quality,
or distribution of assimilates produced in fleshy, acid-accumulating

4

fruit either from light or dark CO^ fixation (Bollard, 1970). The
relative amount of fruit photosynthates partitioned into organic acids
remains unclear.

Two additional approaches to determining the sources of fruit
acid production are made possible by the existence of genetic and
chemical mechanisms which alter fruit acidity. One grapefruit mutant
with only 2/3 the normal acid levels was developed by bud irradiation
of 'Foster' pink grapefruit (Hearn, 1986). Changes in accumulation and
metabolism of organic acid could thus be compared between this
low-acid mutant and a normal-acid counterpart which differ in acid
levels. Arsenate spray is also known to substantially decrease acidity
in grapefruit and is widely used in Florida (Wilson, 1983).

Differences in metabolism and CO^ fixation by normal vs. low acid
grapefruit clones and between arsenated and non-arsenated calamondin
trees may provide information valuable to understanding the control of
acid accumulation in citrus fruit.

The objectives of this research are to quantify the extent to
which fruit organic acids are produced by metabolism of leaf
photosynthates transported to fruit, direct translocation of organic
acids from leaves to fruit, and light and dark C02 assimilation in
fruit during their development. The additional objectives are to
compare these processes as well as activities of acid metabolizing
enzymes in normal- and low-acid grapefruit mutants, and characterize
related effects of arsenate on acid accumulation in citrus fruit.

CHAPTER II
LITERATURE REVIEW

Assimilate Partitioning in Citrus

Assimilate translocation out of leaves has been extensively

studied in many plant species and food crops. The proximity of a given

source leaf to a sink has long been known to affect the extent of

photosynthate export and its subsequent distribution in the plant

(Wardlaw, 1968; Wareing and Patrick, 1975). A greater proportion of

the assimilates from leaves near roots will move below ground, while

photosynthates from upper leaves will be translocated preferentially

to the shoot apex. Assimilates can move either or both directions from

leaves at an intermediate position (Wardlaw, 1968). A similar pattern

of photosynthate distribution was also found in citrus (Powell, 1968;

Kriedemann, 1969a, 1969b, 1970). Assimilates exported from leaves on

the same growth flush with fruit move almost exclusively into these

fruit (Powell, 1968). Leaves farther from fruit, however, export

assimilates in both acropetal and basipetal directions, while
14

C-photosynthates derived from leaves near the soil line show
extensive basipetal movement (Powell, 1968; Kriedemann, 1969a, 1969b;

Powell and Krezdorn, 1977; Brown, 1974).

14

The proportion of C-assirailates received by navel orange fruit
from adjacent leaves also changes seasonally. Variations are
apparently affected by both the position of leaves and competition
between newly developing leaves and fruits (Brown, 1974). Initial

5

6

flushes are the strongest sinks, followed by the developing fruits.
Developing leaves represent the most powerful sinks and do not become
contributing organs until fully mature (Kriedemann, 1969a, 1969b;
Kadoya, 1974). This pattern of assimilate partitioning is different
from that in other fruit crops in which leaves typically export
assimilates when only partially expanded (Bollard, 1970). The
application of gibberellic acid and cytokinin can increase the import
of assimilates or replace the fruit as a sink when fruit is removed
(Powell, 1968; Kriedeman, 1968a; Powell and Krezdorn, 1977; Brown,
1974; Kadoya, 1974). Photosynthate partitioning within- leaves and
allocation in the whole plant can affect the growth of fruits, which
in turn can influence not only the direction and rate of translocation

but also leaf photosynthetic rate (Kadoya, 1974; Lenz, 1979).

14

Transport of C-assimilates into citrus fruit was initially
believed to occur by way of the vascular tissue and hence movement
into juice sacs would be via the vascular bundles on the surface of
the segment epidermis (Kriedemann, 1969a). Anatomical and
radiolabeling studies of grapefruit showed that the majority of
photosynthates translocated into juice sacs moved via dorsal vascular
bundles (Koch, 1984b). The narrow band of distribution for
photosynthates entering citrus fruit from an individual leaf
demonstrates a possible translocational basis for the unequal
distribution of soluble solids in citrus fruit (Koch, 1984a). Sucrose
is apparently the primary transport sugar in citrus phloem

(Kriedemann, 1969a; Koch, 1984b).

14

Export of C-assimilates from citrus leaves can be rapid, but
also may occur for an extended period. Measurable import of

7

C-assimilates into fruits was detected 20 min after exposure of
14

adjacent leaves to C02 (Kriedemann, 1969b, 1970). Still, mature

leaves were able to export labeled photosynthate for at least 40 days
14

after C02 administration (Kriedemann, 1969b, 1970). The majority of
assimilates translocated from leaves to adjacent grapefruit are
transferred within 24 to 30 h, but export appears to occur more
rapidly during greatest rates of fruit expansion (Koch, unpublished
data). Fruit photosynthesis also occurs in citrus but declines toward
maturity (Bean and Todd, 1960; Todd et al. , 1961; Ramakrishnan, 1971;
Moreshet and Green, 1980). Assimilation of C02 per unit area in fruit
is 25-50% of that by leaves in 'Valencia' orange (Moreshet and Green,
1980). The uptake was dependent on fruit size, photosynthetically
active radiation, chlorophyll content, and on different diffusive
conductances of the fruit epidermis (Moreshet and Green, 1980). Fruit
photosynthesis in oranges and lemons contributes little toward fruit
development (Todd et_ al. , 1961; Moreshet and Green, 1980). Similar
results were also found in other fruit crops such as apple, avocado
and mango (Kidd and West, 1947; Pandey and Farmahan, 1977; Chauhan and
Pandey, 1984). Labeled organic acids were detected after ^ C02
fixation in young citrus fruit in light, but the amount was relatively
low compared to the labeled sucrose content (Bean and Todd, 1960). The
extent to which fruit photosynthate is partitioned into organic acids
throughout development remains unclear.

Organic acid production during C02 assimilation in the dark is
well documented in fleshy tissues of desert plants (Edwards and Huber,
1980; Ting, 1985). Uptake of C02 in darkness is essential to
photosynthetic processes in succulent plants which utilize

8

Crassulacean Acid Metabolism (CAM) to conserve water during carbon

assimilation. Dark CO^ fixation is also known to be an anaplerotic

carboxylation reaction in other tissues and is related to pH balance

in cells (Davies, 1973; Bidwell, 1983; Bown, 1985).

Phosphoenolpyruvate carboxylase (PEPc) is the enzyme most commonly

responsible for CC>2 fixation in the dark (Huf faker and Wallace, 1959;

Bown, 1985; Ting, 1985). Data of Bean and Todd (1960) indicated that
14

the C-assimilates formed by citrus fruit in the dark must be
produced within each tissue rather than by extensive translocation
from one to another. More labeled-assimilates were found in pulp of
young intact lemon fruit than other fruit tissues after dark ^C02
fixation. Assimilates were partitioned mainly into organic and amino
acids.

Organic Acids in Citrus Fruits
Organic acids are involved in almost all aspects of plant
metabolism. These have been extensively reviewed in relation to
specific aspects of the metabolism in which they are involved (Thiraann
and Bonner, 1950; Bartholomew and Sinclair 1951; Kefford 1959;
Sinclair, 1961; Beevers et al. , 1966; Kefford and Chandler, 1970;
Ramakrishnan 1971; Ting and Attaway, 1971; Sinclair, 1972; Vandercook,
1977; Sinclair, 1984). Citric and malic acids are respiratory
intermediates. Malate, which is also produced via C02 fixation by PEP
carboxylase, plays an important role in photosynthesis (Edwards and
Huber, 1981; Ting, 1985), pH balance (Davies, 1973; Bown, 1985), and
nitrogen assimilation (Raven and Smith, 1976; Robinson, 1983; Lance
and Rustin, 1984). It can also transport reducing equivalents across

9

membranes (Lance and Rustin, 1984).

Both citric and malic acids are the major organic acids in citrus
fruit, but the type and levels of acids can vary greatly among
different geographic locations and tissues (Sinclair and Eny, 1947;
Ting, 1969; Monselise and Galily 1979). Citrate is the primary acid in
grapefruit juice while malate is the dominant acid in peel (both
flavedo and albedo) (Monselise and Galily, 1979). Acidity is also
unevenly distributed in the juice tissues, and higher levels are found
toward the core region of grapefruit and oranges (Ting, 1969).

Fruit acidity is affected by both genetic and horticultural
factors. Acid content can be as high as 6% (expressed as citric
acid/fresh weight) in lemon fruit or less than 0.1% in acidless orange
or pummelo (Cameron and Soost, 1977; Sinclair, 1984). Fruits produced
on trees with a trifoliate orange rootstock are generally higher in
acid content than those from trees on rough lemon rootstock (Sinclair,
1984). Citrus fruit grown at lower temperatures are higher in acidity
than those produced in warmer climates or during warmer years
(Reuther, 1973). Application of higher levels of nitrogen and
potassium fertilizers also increases fruit acidity (Vandercook, 1977).
In addition, irrigation significantly decreases the acidity in
grapefruit and oranges compared with unirrigated groves (Koo and
McCornack, 1965; Gilfillan et al. , 1976). Arsenate spray about 1 month
after blooming has long been known to decrease acid content in citrus
fruit (Deszyck and Ting, 1958; Wilson, 1983; Rice et al. , 1985). On
the contrary, boron or copper sprays will increase the acidity of
citrus fruit and often have an effect antagonistic to that of arsenate
(Smith, 1973).

10

Organic acids are accumulated throughout fruit development (Ting
and Vines, 1966; Kef ford and Chandler, 1970; Sinclair, 1984), but
timing and rate of accumulation vary with different citrus species.
Acids accumulate more rapidly in young, developing fruit (Erickson,
1968). Total acidity increases during fruit growth; however, the
concentration remains stable or decreases toward maturity (Ting and
Vines, 1966; Erickson, 1968).

Organic Acid Biosynthesis in Citrus Fruits

Reciprocal grafts between sweet and sour lemons have suggested
that the mechanism affecting organic acid accumulation might be
localized primarily, but not entirely in fruit. Sweet lemon fruits
grafted to sour lemon plants remained low in citric acid (Erickson,
1957), but acidity of these fruit was higher than when the same sweet
lemons were grafted to sweet lemon plants. These data indicate that
the leaves and fruit can both affect the acidity of citrus juice
(Erickson, 1957).

Lack of acid accumulation in peels of citrus fruit led Sinclair
and Eny (1947) to suggest that organic acids were probably synthesized
in the juice sacs from carbohydrates. Ramakrishnan (1971) later
proposed that carbohydrates were utilized by a combined operation of
both the glycolytic pathway and TCA cycle, and the citric acid would
therefore be expected to accumulate due to an absence of aconitase
activity. Labeled citrate was found shortly after radioactive
carbohydrates were supplied to fruits (Ulrich, 1970). Citric acid and
to a lesser extent malic acid were the main intermediates accumulated
when the TCA cycle was blocked by different metabolic inhibitors in

11

satsuma mandarin fruit (Kubota and Akao 1973a). Activity of the TCA
cycle was observed in several citrus fruits after addition of labeled
citrate, succinate and pyruvate (Bogin and Erickson, 1965; Kubota and
Akao, 1973a; Akao and Kubota, 1978b). A comparison between the amount
of labeled raalate and citrate formed after adding labeled pyruvate
indicated that more of the pyruvate entering the TCA cycle had arisen
indirectly from CO^ fixation than that which entered via the citrate
synthase reaction (Bogin and Wallace, 1966a, 1967).

Citrate synthase is a major regulatory enzyme in the TCA cycle and
Bruemmer et al. (1977) have suggested that its activity could be
responsible for controlling citric acid production in citrus fruit.
Buslig (1971) indicated that an asynchrony between activities of
citrate synthase and the degradative enzymes, aconitase and isocitrate
dehydrogenase, might explain the accumulation of citrate in citrus
fruit. Bogin and Wallace (1967) proposed that higher citric acid
levels in sour lemon were due to reduction of aconitase activity by
citramalate , an inhibitory acid formed in citrus from pyruvate. The
amount of pyruvate available for citramalate formation would
presumably decrease under circumstances favoring higher C02 fixation
by PEP carboxylase or more rapid conversion of pyruvate to alanine
(Bogin and Wallace, 1967). No further study has been reported on this
hypothesis.

Another contributing factor could be that, as in a series of
experiments with grape (Ruffner, 1982; Ruffner et al. , 1983), the
reduction of acid formation during ripening was found to be due to the
inhibition of glycolytic carbon flow. This in turn resulted in
accumulation of imported sugars. At the same time, the ripening fruit

12

could transform acids into sugars by reactions involving a reversal of
the glycolytic pathway (Ruffner et al. , 1975).

Although organic acids are minor components in phloem sap, most
organic acids such as citric, malic, oxaloacetic and tartaric acids
found in fruit have been detected in exudates from phloem. The type of
organic acids varies with plant species (Ziegler 1975). Malate in
phloem of Ricinus plants is believed to be associated with the
maintenance of ionic balance (Hall and Baker, 1972). Also, the direct
translocation of organic acids from leaves has been demonstrated in
grape (as reviewed by Ulrich, 1970). The contribution of organic acids
translocated from leaves or roots to acid accumulation in citrus fruit
is unknown.

The role of dark CO^ fixation in organic acid biosynthesis has

been debated since Huf faker and Wallace (1959) first suggested that it

might be associated with the accumulation of acid in citrus fruit.

Subsequent studies identified labeled organic acids in young lemon
14

fruit after dark C02 fixation (Bean and Todd, 1960). However, the
amount of dark C02 fixation did not correlate with the acidity of
fruit tissues when different species, fruit parts, and methods were
used (Clark and Wallace, 1963; Bogin and Wallace, 1966a). Clark and
Wallace (1963) found sweet lime, which was lowest in acid content, had
the highest C02 fixation rate in juice vesicle homogenates. Bogin and
Wallace (1966a) found that C02 fixation was greater in sweet lemon
extracts when crude PEP carboxylase, malic enzyme and isocitrate
dehydrogenase were used, whereas maximal rates were observed in sour
lemon when entire peeled fruit was used. In Citrus natsudaidai , either

in light or dark, the amount of C02 fixation in sweet fruit juice

13

vesicles was greater than in sour fruit (Akao and Tsukahara, 1979). In
contrast, CO^ fixation in juice vesicles was lower in arsenate treated
fruit (Akao and Kubota, 1978a). Despite differing results, it was
agreed that dark CO^ fixation was greater during the period of rapid
organic acid accumulation in fruit and declined toward maturity, as in
grape berry (Kriederaann, 1968a).

Further research on different citrus fruit showed that organic
acids (mainly citric and malic) were labeled after dark C02 fixation
(Bean and Todd, 1960; Young and Biale, 1968; Akao and Kubota, 1978b;
Akao and Tsukahara, 1980a, b). Isolated juice vesicles were found to

14

accumulate more C-assimilates than flavedo or albedo during dark
14

C02 fixation. Also, more labeled citrate and malate were found in
juice vesicles than in albedo and flavedo (Bean and Todd, 1960).

14

Products of C02 assimilation in the dark seemed to be formed within
each tissue rather than by extensive translocation from one tissue to
another (Bean and Todd, 1960).

Arsenate Treatment in Citrus

Asenate sprays have been applied to Florida grapefruit trees for
many years to decrease total acidity in juice vesicles at harvest
(Miller et al. , 1933; Deszyck and Ting, 1958, 1960; Wilson, 1983). The
most effective application period is 1 to 6 weeks following bloom
(Wilson, 1983). A similar effect has been identified in other citrus
species such as orange, satsuma mandarin, and calamondin (Buslig,

1971; Akao and Kubota, 1978a; Wilson, 1983). The response to nonleaded
arsenate was similar to those of lead arsenate indicating that arsenic
was the major contributor to acidity reduction (Rice et al. , 1985).

14

Most organic and inorganic arsenical compounds will cause similar
effects (Wilson, 1983). In a whole tree, only sprayed portions are
affected, suggesting the action of arsenate sprays is not broadly
systemic. The response of actively growing leaves to arsenate spray
was evident almost immediately in fruit and the effect persisted for
about 18 months (Miller et al. , 1933).

High levels of arsenic are toxic to both plants and animals,
despite the role of arsenate as an essential nutrient for certain
animal species (Wilson, 1983). Arsenate is known to uncouple
phosphorylation and is used as a herbicide due to its influence on
sulfhydry 1-group enzymes (Vines and Oberbacher, 1965; Beevers et al. ,
1966; Knowles, 1982). Oxidation of pyruvate and <<-ketoglutarate , both
respiratory intermediates, is also influenced by arsenate (Beevers et
al. , 1966). Aerobic fermentation, as evidenced by greater CO^
evolution and alcohol accumulation, was induced by arsenate treatment
(Beevers et al. , 1966). Vines and Oberbacher (1965) proposed that
this uncoupling effect was the primary mechanism of acidity reduction
in citrus fruit. Akao and Kubota (1978a) determined that the
fixation rate of juice vesicles from fruit sprayed with lead arsenate
was lower than that of vesicles from unsprayed fruit. Similar results
were also obtained in citrus juice tissues by Huffaker and Wallace
(1959), but the activities of PEP carboxylase and malic enzyme in
citrus leaves and fruit albedo were not affected or were only slightly
increased. However, arsenic residue in juice sacs of sprayed fruit is
usually negligible and normally not significantly different from that
of unsprayed controls (Gilf illan et aU , 1976; Rice et al. , 1985).

Lack of either lead or arsenate in juice sacs after postbloom sprays

15

led Rice al. (1935) to conclude that the primary effects of
arsenate spray might be elicited in the leaves. Gilfillan et_ al.

(1976) compared the arsenic concentration in peel and pulp and found
significantly higher arsenic levels in peel after treatment. The
possible effects of higher arsenic in peel thus cannot be excluded.

Arsenate has long been known to increase the respiration of leaves
in grapefruit. Decreased acidity was supposed to be associated with
the increase of leaf respiration (Miller et al. , 1933). Experiments on
isolated pea chloroplasts indicated that arsenite affected light
modulation and photosynthetic induction through its inhibition of
light activation of enzymes in the reductive pentose phosphate (C^)
cycle (Marques and Anderson, 1986).

Boron has been reported to increase fruit acidity and show an
antagonistic effect to arsenate. The overall antagonism was
essentially the same when both elements were included in the same
spray, applied in separate sprays, separated in a time interval, or
when boron was applied to soil and arsenate to the foliage (Smith,
1973). However, Miller e_t a_l . (1933) indicated that soil application
of arsenate was ineffectual in reduction of grapefruit acidity. The
mechanism by which arsenate decreases acid levels in citrus fruit is
unknown.

Enzymes Associated with Organic Acid Metabolism
Several enzymes are related to organic acid synthesis and
degradation. They may be divided into two groups according to their
functions and locations. Enzymes of malate synthesis are located in
cytoplasm and mitochondria, while enzymes of citrate synthesis are

16

located mainly in the mitochondria.

Malate Synthesis from PEP

Three enzymes, PEP carboxylase (PEPc) , malate dehydrogenase (MDH)
and NAD-malic enzyme (NAD-ME) regulate the synthesis and oxidation of
malate, which functions as a source of carbon for anaplerotic
operation of the citric acid cycle (Lance and Rustin, 1984; Naik and
Nicholas, 1986). Latzko and Kelly (1983) list eleven physiological
functions dependent on the concentration of malate and the associated
relationship between activity of PEPc and MDH.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) is almost ubiquitous
in plants and plays a particularly important role in carbon fixation
by C4 and CAM plants (O'Leary, 1982). It is located in cytoplasm
(Edwards and Huber, 1981) and catalyzes the reaction
PEP + HC03~ > OAA + Pi

Normally, OAA is rapidly converted to malate in most plant systems. At
least four different forms of PEP carboxylase exist in higher plants
and are believed to be associated with the specific functions among
plants. A PEP carboxylase from non-green root tissue was found to have
a K and V considerably lower than that of the enzyme from leaves
of CAM and C^ plants (Ting and Osmond, 1973). Even in extracts from
the same tissue, multiple forms of PEPc may exist that contribute to a
diurnal variation in overall activity (O'Leary, 1982). The PEPc-MDH-ME
enzymes also act as a cellular pH stat in plants (Davies, 1973).
Concentration of malic acid in the cytoplasm depends on the balance
between carboxylation (by PEPc) and decarboxylation (by ME).
Phosphoenolpyruvate carboxylase has maximum activity near pH 8, while
malic enzyme has maximum activity close to pH 6.5. When cellular pH

17

rises, PEPc is active and ME less so. At the same time, raalate
accumulates and releases two protons for each malate, thus balancing
the increase of OH ions (Davies, 1973). Phosphoenolpyruvate activity
was demonstrated in leaves, roots, fruit flavedo and juice vesicles of
'Valencia' orange (Huffaker and Wallace 1959). Huffaker and Wallace
(1959) also proposed a pathway of citric acid synthesis in citrus
fruit vesicles via a sequence of PEPc-MDH-citrate synthase. Later
experiments in the same laboratory confirmed that dark CO^ fixation
rates were higher during the period of maximal acid accumulation.
However, CO^ fixation was also greater in low acid fruit, which is
inconsistent with their hypothesis (Clark and Wallace, 1963; Bogin and
Wallace, 1966a).

Malate dehydrogenase is found in chloroplasts , mitochondria,
cytoplasm and microbodies, and catalyzes the reaction

OAA + NADH(NADPH) < > malate + NAD(NADP)

The chloroplast enzyme is specific for NADP+, the others are specific
for NAD+. Vine (1968) identified two forms of malate
dehydrogenase (MDH) isolated separately from mitochondria and
cytoplasm of grapefruit juice vesicles. The mitochondrial MDH was
inhibited by elevated concentrations of oxaloacetic acid while the
cytoplasmic MDH was insensitive. The enzymes of both sources were
specific for L-malate and insensitive to D-malate.

Malic enzyme catalyzes the reaction

Malate + NAP(NADP) > pyruvate + C0? + NADH(NADPH)

Malic enzyme, like MDH, has many isozymes which are distributed in
chloroplasts, mitochondria, and the cytoplasm, but the cytosolic and
chloroplastic ME are specific for NADP+, while the mitochondrial ME is

18

specific for NAD (Edwards and Huber, 1981; Lance and Rustin, 1984).
One of the differences between plant and animal respiration is that
plants can form pyruvate and citrate from malate alone, through the
combined action of MDH and malic enzyme (Palmer, 1976; Moller and
Palmer, 1984). This mechanism allows the interconversion of all TCA
cycle intermediates, thus the input of one intermediate can supplement
the pool of any other intermediate (Moller and Palmer, 1984).

Formation of pyruvate from PEP in plants is believed to occur to a
greater extent via PEP carboxylase, MDH, and malic enzyme than through
pyruvate kinase (Bogin and Wallace, 1966a; Lance and Rustin, 1984).
Also, evidence indicates pyruvate is not effectively transported
across the mitochondrial membrane in plants (Wiskish, 1980).
Biosynthesis and oxidation of malate are regulated by NADH, OAA, pH,
and adenylate balance of the cell. The control of MDH, ME and
regulation of the malate/OAA concentration in the cell, as well as its
relationship to cell energy charge and nitrogen assimilation have been
reviewed by Wiskich (1980), Palmer (1984), Lance and Rustin (1984) and
Naik and Nicholas (1986).

Citrate Synthesis and Subsequent Citrate Degradation in the TCA Cycle
Citrate synthase (EC 4. 1.3.7, or citrate condensing enzyme,
condensing enzyme, citragenase, OAA transacetase) is located in
mitochondria and glyoxysomes and catalyzes the reaction

OAA + acetyl-CoA + H^O > citrate + CoA

Aconitase (EC 4. 2. 1.3) catalyzes the interconversion of citrate,
aconitate and isocitrate

citrate < > cis-aconitate < > isocitrate

The equilibrium constant favors the formation of citrate (Moller and

19

Palmer, 1984). Many reviews of citrate synthase and reactions
associated with citrate have been presented (Srere, 1972; Srere, 1975;
Weitzman and Danson, 1975; Wiskich, 1980; Moller and Palmer, 1984).
Overwhelming data on this TCA cycle enzyme have been accumulated. Only
the information related to organic acid accumulation in citrus fruit
will be cited here.

Citrate synthase was found in leaves, peel and pulp of orange and
lemon (Srere and Senkin 1966), grapefruit (Ting and Attaway, 1971) and
satsuma mandarin (Hirai and Ueno 1977). Peel and pulp of green oranges
were found to contain more of this enzyme than respective tissues from
mature fruit. Activity of citrate synthase was also higher in peel
than pulp (Srere and Senkin 1966).

An imbalance between activity of citrate synthase and subsequent
TCA cycle enzymes, aconitase and isocitrate dehydrogenase, has been
proposed to be responsible for the citrate accumulation in citrus
fruit (Varma and Ramakrishnan, 1956; Raraakrishnan and Varma, 1959;
Ramkrishnan, 1971; Buslig, 1971; Bruemmer et al. , 1977). Far less is
known about aconitase than citrate synthase. Palmityl CoA and ATP
reportedly decrease the affinity of citrate synthase for oxaloacetate
making the other enzyme more competive. The NADH is a negative
effector of malate dehydrogenase and a potential regulator of citrate
synthesis (Bogin and Wallace, 1966b; Bruemmer et al. , 1977).
Phosphoenolpyruvate carboxylase catalyzes an irreversible reaction;
therefore it does not compete with citrate synthase and MDH for
available oxaloacetate (Bruemmer et^ £l . , 1977). Citrate synthase
activity increases, and aconitase and isocitrate dehydrogenase
decrease, during the rise in acid content of lemons from 0.9% to 4.0 %

20

(Ramakrishnan and Varma, 1959).

Data of Vines and Metcalf (1967) indicated that variation in
mitochondrial activity in grapefruit coincided with the rise and fall
of total juice acidity, suggesting the possibility that changes in
citric acid content may be correlated with the general phosphorylative
ability of the fruit mitochondria. Hirai and Ueno (1977) followed the
enzymatic changes of satsuma fruit during development and found that
the ratio of phosphoenolpyruvate caboxylase and NAD-dependent
isocitrate dehydrogenase was highest during most rapid acid
accumulation. Isocitrate dehydrogenase may play an additional
regulatory role in acid accumulation.

CHAPTER III

DISTRIBUTION OF ASSIMILATES PRODUCED DURING
LIGHT AND DARK 14C0 FIXATION BY DEVELOPING
GRAPEFRUIT AND ADJACENT SOURCE LEAVES

Introduction

The vast majority of assimilates are produced by leaves during
photosynthesis in light, but CO2 fixation occurs in fruits as well as
leaves, and in both light and dark. Partitioning and translocation of
photosynthates derived from source leaves of whole plants is
relatively well-documented in a number of commercially important food
species (Wardlaw, 1968; Wareing and Patrick, 1975); Less information
is available for fleshy fruit crops, including citrus, but these fruit
are known to be strong sinks for leaf photosynthates (Kriedemann,
1969a, 1969b; Kadoya, 1974). The presence of fruit can affect
photosynthetic efficiency of citrus leaves and partitioning of
photosynthates (Lenz, 1979). Assimilates are also preferentially
translocated from citrus source leaves to aligned sections of fruit
tissue (Koch and Avigne, 1984). In addition, photosynthates from
leaves enter juice sacs very slowly, via a limited number of vascular
bundles and extensive phloem-free areas of the transport path (Koch,
1984b; Koch ejt a_l . , 1986). Seasonal alternations have been reported by
Brown (1974) for leaf-derived assimilates arriving in navel oranges.
Changes which occur in photosynthate translocation and distribution
within these fruit have not been examined during their development,

21

22

however.

Photosynthesis also occurs in fruit of many crops. Studies of pea
and soybean indicated that the pods could provide photosynthates to
developing seeds by reassirailation of respiratory CC>2 (Quebedeaux and
Chollet, 1975; Atkins et al^. , 1977). In a young green apple or citrus
fruit, photosynthetic CO^ fixation is of sufficient magnitude to
conserve 20-80% of the CC^ release by dark respiration depending on
stage of development (Kidd and West, 1947; Bean et al. , 1963; Shaedle,
1975).

Fruit photosynthesis was reported to decrease toward maturity in

citrus (Bean and Todd, 1960; Todd et al. , 1961; Ramakrishnan, 1971;

Moreshet and Green, 1980). Contribution of fruit photosynthesis to

orange and lemon growth is believed to be relatively small compared

with that of assimilates translocated from leaves (Todd et al. , 1961;

Moreshet and Green, 1980). However, about 25% of the
14„ u

C-photosynthates remain in satsuraa mandarin juice tissues 5 days

14

after fruit exposure to CO2 in the light (Akao and Tsukahara,

1980a). Little information is available, however, on the partitioning,
distribution, or redistribution of photosynthates produced by
grapefruit or other fleshy fruit.

Dark CO2 fixation, an anaplerotic reaction contributing to amino
and organic acid formation (Bidwell, 1983), is involved in root growth
(Splittstoesser , 1966), and flower bud development of 'Valencia'
oranges (Vu ejt al_. , 1985). A similar relationship may exist for
organic acid synthesis in citrus juice tissues. Minimal research has
been directed toward analysis of assimilates arising from
non-photosynthetic carboxylation reactions in fruit. However, greater

23

amounts of C-assimilates were found in citrus juice sacs than

14

surrounding tissues after each was exposed to CC^ in the dark (Bean
and Todd, 1960). Greater proportions of labeled organic acids
(predominantly in citrate) and amino acids were also recovered in
juice tissues after dark CO^ fixation by fruit than in light. This
process has long been considered one possible source of organic acid
synthesis in citrus (Huf faker and Wallace, 1959; Bean and Todd, 1960;
Young and Bial, 1968). The extent of dark CO^ fixation in whole fruit
has not been examined, nor have developmental changes in these
reactions.

One purpose of the present work is to compare the distribution of

14

labeled assimilates derived from fixation in light and dark by

fruit and adjacent source leaves throughout grapefruit development.

The second is to assess the relative contributions of each assimilate
source to a given stage of fruit growth.

Materials and Methods

Fruits and/or branches with source leaves adjacent the fruit were
randomly selected from between 1 and 2 m above ground level on the
exterior canopy of 4-year-old grapefruit trees ('Foster Pink' seedless
clone # 1-26-39) on Savage sour orange or sour orange rootstocks in
Lake Wales, FL (Hearn, 1986). No significant differences were evident
in quality of fruit from trees on these two rootstocks (data not
shown). Samples from a given tree were used for different analyses.
Each tree represents one replication and four trees were used for each
experiment. Fruit were harvested on May 7, June 25, July 27 and
September 10, 1986 (at about 40-day intervals), after 2, 3.5, 5, and

24

6.5 months' growth, respectively. Pedicels and branch bases were

immediately immersed in water. At each developmental stage, detached

fruit or individual leaves adjacent to fruit were enclosed in a

cuvette and exposed to 13.6 pCi 1_1 14CC>2 for 1 h in light followed by

ambient air for 24h (12h:12h light:dark photoperiod). Levels of CC>2

were monitored during studies using an infrared gas analyzer (IRGA)

(Model 865, Beckman Instruments Inc., Fullerton, CA) and a closed,

flow-through system. Data were used to calculate mean specific
... 14

activities of CC>2 during periods when respiratory CC>2 was increasing

total amounts present. Temperature was maintained at 28°C and

photosynthetic photon flux density at 800 to 1000 pmol m~2 s_1. Light

was provided by a Lucalox lamp (General Electric Co., Cleveland, OH).

Similar experiments were conducted in 24 h of continuous darkness

using cuvettes completely sealed with several layers of foil. Fruit
14

were exposed to C02 within a few hours after pedicels or branches
were removed from trees, and studies with source leaves were conducted
the following morning.

Experiments were terminated by separating tissues into leaves and
pedicels (if present), flavedo (outer, pigmented peel), albedo (inner,
non-pigmented peel), and juice tissues. After some *4C02 treatments,
fruit were halved longitudinally to compare the effect of leaf
location or light source position on assimilate distribution in the
fruit. Samples were frozen in liquid nitrogen, and extracted in 80%
(v/v) ethanol. The ethanol-insoluble fraction was oven-dried for 4
days at 60 C. Radioactivity in each sample was quantified by liquid
scintilation spectrometry (LKB , Gaithersburg, MD). The ethanol-soluble
fraction was measured by pipetting 0.1 ml of sample into 15 ml

25

scintilation cocktail (Scinti Verse II, Fisher Scientific CO., Fair
Lawn, N J ) . The ethanol- insoluble fraction was measured by suspending a
known portion of the sample in scintilation cocktail.

Results and Discussion

Fruit dry weight increased continuously during the developmental
period examined, but did so most markedly during the 3rd and 4th
months of growth (May to June) (Fig. 3-1A). Sink strength of fruit for
photosynthates therefore was also greater at this time. Flavedo and
albedo of peel accumulated the majority of their dry weight during
this period and added only minute amounts at later stages. Increases
in dry weight of juice tissues accounted for almost all change in
total fruit dry weight during the second half of fruit growth and
presumably also fruit demand for photosynthates.

Fresh weight gains during development (Fig. 3-lB) were
proportional to those of dry weight, but more pronounced in juice
tissues. Both fresh and dry weight followed trends similar to those
observed in other citrus fruits (Bain, 1958; Sinclair , 1984 ) . The rapid
increase in fresh and dry weight of fruit was typical of Stage II
development observed in grapefruit (Lowell, 1986) and 'Valencia'
oranges (Bain, 1958).

Sources of dry matter increase by grapefruit were compared
throughout fruit development by examining ^C-assimilate recovery in
fruit 24 h after 1 h of light or dark CO^ fixation by either leaves
or fruit (Table 3-1). The total dpra recovered from all tissues after
photosynthesis by a single leaf, as well as the percentage
translocated to fruit, was by far the greatest at early stages of

Fig. 3-1. Developmental changes in dry weight (A) and fresh weight
(B) of tissues in grapefruit ('Foster' pink seedless clone // 1-26-39).
Vertical bars denote the SE of 4 replications, but are present only
where larger than tissue/fruit symbols.

27

28

Table 3-1. The total C contribution and C-assimilate recovery in
grapefruit ('F^ter' pink seedless clone // 1-26-39) 24 h after 1 h of
light or dark CO |£xation by either fruit, or an adjacent source
leaf. Exposure to CO, (13.6 uCi 1 ) was followed by ambient air

for 24h either in a 12:12 light: dark period or in continuous dark.

Date

A single source leaf

Detached

fruit

L1§ht

Dark

Light

Dark

(dpm fruit

1 x 108)

May 7

2.74 + 0.75y

0.006 + 0.002

1.41 + 0.34

0.026

+

0.001

June 25

0.84 + 0.27

ND

1.49 + 0.22

0.032

+

0.004

July 27

0.41 + 0.12

ND

2.81 + 0.82

0.067

+

0.010

Sept 10

0.26 + 0.11

ND

0.75 + 0.20

0.051

+

0.010

(jiraol C f

. -1 Nx
ruit )

May 7

8.78 + 2.33

0.020 + 0.006

10.78 + 2.62

0.53

+

0.02

June 25

2.64 + 0.88

ND

15.89 + 2.35

0.64

+

0.08

July 27

1.29 + 0.35

ND

36.51 +11.21

0.99

+

0.14

Sept 10

0.87 + 0.37

ND

14.03 + 3.49

1.97

+

0.51

(dpm g * fruit

4

dry wt x 10 )

May 7

369.7+ 100.9

0.77 + 0.11

179.3 + 40.4

2.70

+

0.28

June 25

26.4+ 10.0

ND

43.5 + 5. 1

1.03

+

0.07

July 27

9.4+ 3.2

ND

60.0 + 16.6

1.50

+

0.17

Sept 10

4.5+ 1.7

ND

14.6 + 3.5

0.88

+

0. 13

z _
Fruit

were harvested

on May 7, June

25, July 27 and

Sept.

10

i, 1986,

when approximately 2, 3.5,^, and 6.5 months old, respectively. The
amount recovered from dark CO fixation in leaves was not
detectable after June.

y

Values are means of 4 replications +_ SE. Values for source leaves in
the light are amounts of leaf C-photosynthates translocated to
fruit, representing 58.11 + 4.48%, 41.77 + 10.56%, 29.16 + 8.99%,
and 32.95 +_ 4.80% of total dpra recovered for May, June, July, and
September, respectively.

The C contributions were calculated based on measurements of
changing specific activity during experiments (see text), and on
expected differences in numbers of carbon atoms labeled in products
of photosynthesis vs dark ^CO^ fixation.

29

fruit development, when dry weight was increasing most rapidly (Table

3-1). This is consistent with observations in citrus (Kadoya, 1974;

Lenz, 1979) and numerous other species (Wardlaw, 1968; Bollard, 1970;

Daie, 1985) which show that photosynthetic rates are often greater

when fruit are present. In addition, greater proportions of

photosynthetic products are typically exported when demands for them

are high (Wardlaw, 1968; Bollard, 1970; Daie, 1985).

14

Fruit CO2 fixation in the light increased from May to July, as

fruit surface area expanded, but decreased thereafter (Table 3-1).

Reductions in fruit photosynthesis at later stages of development have

also been identified in other citrus fruit (Bean and Todd, 1960;

Moreshet and Green, 1980), but the drop observed here substantially

preceded color break. Total pmol C derived from 1 h of dark fixation
12 14

in CO^ + CO2 by fruit increased during development, and probably

resulted from the larger fruit size.

14

In all CO2 assimilation experiments, greatest total dpm per gram
fruit dry weight was recovered from very young grapefruit in May.
Values gradually decreased during fruit development (Table 3-1).

The relative C contributed to fruit from 1 h CO2 fixation by
various sources was further compared using parameters described in
Table 3-2. Calculations included jumol C recovered in fruit, time, bias
correction, and the numbers of leaves required to support fruit
growth. Results showed that leaves were the primary C source for fruit
growth throughout development (Table 3-3). Fruit photosynthesis was
also an important source of assimilates (18.9% to 47.3%) after the
majority of expansion had occurred. Dark CC^ fixation by fruit
provided only a small amount of C to fruit growth throughout the fruit

30

Table. 3 2. Parameters utilized for assessments of relative C
contributions by various sources to growth of grapefruit.

Sources

umol C recovered
in fruit

Time

(12h)Z

Bias

correction^

# of leaves Sum

or fruits x

Leafw

(Light)

LL

1

1 to 2

30 to 60 30 to 12(

FruitV

(Light)

FL

1

0.1 to 1

1 0. 1 to 1

FruitU

(Dark)

FD

2

1

1 2

Unpublished data (Tomlinson and Koch, 1987) indicate "dark" CO
fixation by PEP carboxylase continues in juice sacs in the ligflt.

y

C02 level decreased slowly during the 60 min exposure time due to
the large volume of the system relative to leaf size. However, CO
levels during some experiments dropped to approximately one half^
the initial level, potentially decreasing total C0„ assimilation for
the period by the same amount. Light i4C02 fixation by fruit took
place in C02 levels 8 times greater than ambient air only during May
when respiratory rates were very high (not shown).

Research of Shamel and Pomeroy (1934) indicate a minimum of 60
leaves are required to support a single grapefruit, but assimilates
from these leaves were also allocated to other parts of the tree.
Data in the present study show that demand by fruit would be maximal
only during early fruit development when rates of growth were
greatest.

w

Relative contribution of leaf = (30 to 120) x LL

photosynthesis to fruit (30 to 120) LL + FL + 2 FD

Relative contribution of fruit
photosynthesis to fruit

FL

(30 to 120) LL + FL + 2FD

Relative contribution of dark = 2 FD

fruit C02 assimilation to fruit (30 to 120) LL + FL + 2 FD

31

Table 3-3. The relative overall C contribution by various sources to
growth of grapefruit.

Time

Source

Light

leaves

Dark

Fruit2

Light

Dark

(%

of overall C

contribution)

May

95.7

to

98.9

0.01

1.0 to 3.9

0.1

to

0.4

June

82.2

to

94.9

NDy

4.8 to 16.5

0.4

to

1.3

July

50.1

to

80.1

ND

18.9 to 47.3

1.0

to

2.6

Sept

59.2

to

85.3

ND

11.5 to 31.8

3.2

to

8.9

Values for CO^ fixation by fruit should be considered minimal
estimates due to the higher internal CO^ concentration and
extensive refixation of respiratory CC^*

not detectable at later stages.

32

development. Kriederaann (1968c) suggested that dark CO^ fixation by
apricots could continue contributing to growth of their flesh after
fruit chlorophyll content had decreased. Although carbon from 1 h of
dark CO^ fixation by grapefruit increased considerably at later stages
of fruit development. It reached a maximum estimate of only 3.2% to
8.9% of total C recovered from various sources (Table 3-3). The
suggestion by Bidwell (1983) that dark CO^ fixation may be an
anaplerotic reaction in plant tissues, is consistent with the high
percentage recovered in grapefruit 24 h later as organic and amino
acids (about 40% for each).

Data in Fig. 3-2 show that in May, when young fruit were

accumulating dry weight most rapidly, about 58% of the total
14

C-photosynthates from adjacent source leaves was transported into

14

fruit within 24 h. The percent of C-photosynthates exported from
leaves to fruit decreased during fruit development, as did the overall
rate of dry weight increase by fruit. Results are consistent with
previous studies which indicate that the presence of fruit can
increase the proportion of the photosynthates exported (Bollard, 1970;
Kadoya, 1974; Lenz, 1979). The stage of calaraondin fruit development
has also been found to influence overall allocation of photosynthates
within trees (Lenz, 1979).

14

Distribution of leaf-derived C-photosynthates within fruit

appeared to change during development in approximate relation to the

extent of new dry matter added to individual tissue. Still, juice

14

tissues and albedo shared most of the C-assimilates imported from
leaves in May, despite nearly equal dry weight gains by flavedo,
albedo and juice tissues. In addition, new dry weight added to juice

|£g* 3-2. Developmental changes in partitioning and distribution of
C-assimilates in grapefruit ('Foster' pink seedless clone # 1-26-39)
after ex^sure of either intact detached fruit or an adjacent source
leaf to C02 for 1 h and ambient air for 24 h in either a 12:12
light: dark regime or continuous dark. Shaded ( ■ ) and open ( a )
portions of columns indicate respective partitioning between soluble
and insoluble (starch and/or cell wall constituents) fractions within
a given tissue. Vertical bars at the tops of columns denote the SE of
4 replications for tissue totals. Ped=pedicel, Fl=flavedo, Al=albedo,
Jt=juice tissues.

34

80

40

-A. May 7

(ca 2 mo.)

Leaf

TJ

Q)

a?

>

o

o

Q)

c o
<D

ca

s:

•*->

c

>

CO

o
*— >

0

SI

Q.

1

O

ca

o

80

40

-B. June 25

(ca 3.5 mo.)

- T

80

40

O 80

40

Source leaf

Light

Green fruit

Light

Dark

T

-r

■ . ’

—

Ilfo

_

'

-• 5 ,•

■

-C. July 27

(ca 5 mo.)

T

-T-

m!

:S'£-

80

40

80

40

80

40

80

40

Leaf Ped FI Al Jt FI Al Jt FI Al Jt

% of total fruit 14 C-assimilates recovered

35

tissues demonstrated that they were by far the dominant sink for

photosynthates in fruit after June, yet the proportion of leaf
14„ . ...

C-assimilates recovered in juice tissues was greater still. The

basis for this difference may have been gain in long-term loss of
14

C-photosynthates from peel either through transfer to other tissues

or via greater release of respiratory 1^C02»

14

Translocation and distribution of C-assimilates resulting from
14

CC>2 fixation by leaves in the dark was similar to that in light,

but a greater percentage (about 80%) was retained in leaves.

14

Distribution of C-assirailates within fruit differed depending on
14

the site of CO2 fixation, its occurrence in light vs. dark, and the

stage of fruit development (Fig. 3-2).

Flavedo appears to be able to fix C02 either from the external

environment or from endogenous respiration (Bean et al. , 1963).

Photosynthetic refixation of respiratory CO2 by grapefruit was

indicated in the present study by a comparison between CO2 exchange in

light and dark (data not shown). However, 24 h after photosynthesis by

intact fruit, the photoassimilates were shown here to remain

predominantly in the peel (either pigmented flavedo or white albedo)

14

(Fig. 3-2). More than 60% of the C-photosynthates remained in

14

flavedo 24 h after fruit were exposed to CO2 in the light. Less than
14

30% of the total C-photosynthates moved inward to the albedo, and
fewer than 4% were recovered in juice tissues. This is raarkly
different from the situation in soybean and pea, where conservation of
respiratory CO2 contributes to seed growth (Quebedeaux and Chollet,

1975; Atkins et al. , 1977).

14 14

Distribution of C-assimilates in fruit after dark CO2 fixation

36

differed substantially from that of photosynthesis in the light (Fig.

3-2). A considerably greater portion of the assimilates labeled during

dark. CC^ fixation were localized in juice tissues, and in several

instances also the albedo. More radioactivity was recovered in juice

tissues than any other fruit part, except in May when juice sacs made

up the smallest portion of the fruit. The total percentage of
14

C-assimilates in juice tissues increased dramatically after June and
remained constant at later stages.

Dark CC^ fixation can be an anaplerotic reaction for amino and
organic acid synthesis and is often associated with growth, pH
balance, and energy supply in plants (Latzko and Kelly, 1983).
Assimilation of CO 2 by fruit observed here may also be related to the
organic acid synthesis in citrus fruits as proposed by Huffaker and
Wallace (1959). The developmental timing and extent of dark CO2
fixation in the present study are consistent with both of the above.
The role of dark CO^ fixation in organic acid synthesis will be

examined further in subsequent chapters.

14

A greater portion of fruit C-photosynthates were generally

recovered in the ethanol— soluble fraction (primarily sugars, organic

acids, amino acids and essential oils) than in the ethanol-insoluble

fraction (primarily starch and structural components) (Fig. 3-2).

Radioactivity in the ethanol-insoluble fraction was greatest early in

fruit development, and might be associated with the rapid increase in

dry weight of cell wall constituents during this period. The ratio of
14„ ...

C-assimilates incorporated into the alcohol-soluble and insoluble
fractions of flavedo, albedo and juice tissues were approximately
similar to the total proportions of those constituents in the tissues

37

Table 3-4. Distribution of ethanol-soluble C-assimilates in fruit
tissues after 1 h of photosynthesis by intact fruit in * CO , and 24
additional h in ambient air. ^

Treatments

z

% of ethanol-soluble
14C-photosynthates recovered^

Total dpm
recovered

Flavedo

Albedo

Juice

tissues

dpm x 10^

June

lh

92.5

+

1.4

6.0

+

1.5

1.5

+

0.2

2. 17

+ 0.33

lh +

24h

64.6

+

3.1

27.2

+

2.4

8.2

+

0.9

0.98

+ 0.47

July

lh

96.0

+

2. 1

2.8

+

1.4

1.2

+

0.7

3. 14

+ 3.36

lh +

24h

69.7

+

0.5

23.4

+

0.8

6.9

+

0.6

4.74

+ 1.64

Sept

lh

95.0

+

0.5

3.9

+

0.4

1.1

+

0.1

3.57

+ 0.55

lh +

24h

75.7

+

1.0

22.3

+

1.0

2.0

+

0.2

2.73

+ 0.58

Fruit harvested on June 7, July 8, and Sept. 10, 1985, after
approximately 3, 4, 6 months' growth, respectively.

y

Value are means of 4 replications + SE.

38

(Sinclair and Jolliffe, 1960).

Some redistribution of fruit ^C-assimilates from flavedo to

interior tissues was indicated by a comparison between distribution 1

h and 24 h after fruit photosynthesis in 14C0 fixation by fruit in

light (Table 3-4). Percentages of labeled ethanol-soluble

photosynthate recovered in albedo and juice tissues of intact fruit

were minimal after 1 h (means of 4.2 % and 1.3 %, respectively),

suggesting that little inward movement of labeled assimilates had

occurred during this time and/or that rates of exogenous 14C02

fixation by inner tissues were low. Portions transferred to albedo

during the subsequent 24 h were considerably greater than those moving

into juice tissues. Approximately one quarter of the total was

recovered from the inner peel, while, only 0.7% to 2.4 % of the fruit
14

C-photosynthates had moved into juice tissues after 24 h (Fig. 3-3).

14

Even 7 days after CO2 administration to fruit, a mean of less

than 4 / of labeled fruit photosynthates had moved inward to juice

sacs (Fig. 3-3). Photosynthesis in fruit also contributed minimally to

total carbon balance of juice tissues in lemon and orange (Todd et

, 1961; Moreshet and Green, 1980). Results here also support this

conclusion for grapefruit juice sacs, but data from other citrus

differ. In calaraondin fruit, for example, about 23% of fruit
14„ u

C-photosynthates were recovered in juice tissues 7 days after
exposure of intact fruit to CO^ (Chapter V). In satsuma mandarin 25%
to 26% of photosynthates labeled by fruit were localized in juice
tissues 5 days after exposure to ^C02 (Akao and Tsukahara, 1980a).

The contrasting results may be due to structural differences between
peel of calamondin, satsuma mandarin and grapefruit, because albedo of

Fig. 3-3. Partitioning and distribution of C-photosynthates after
exposure of intact, recently-^atached grapefruit ('Foster' pink
seedless clone # 1-26-39) to CO- for 1 h followed by ambient air for
1 day (A) or 7 days (B) in a 12 h light: 12 h dark regime. Shaded
( H ) and open ( □ ) portions of columns indicate respective

partitioning between soluble and insoluble (starch and/or cell wall
constituents) fractions within a given tissue. Vertical bars at the
tops of columns denote the SE of 4 replicatios for tissue totals, and
are too small to be pictured where not shown. Similar designations
within columns represent the SE for component fractions of each total.
Fl=flavedo, Al=albedo, Jt=juice tissues. Total dpm recovered after 1
day and 7 days were 1.41 (+0.34) X 10° and 0.39 (+0.04) X 10°,
respectively, equivalent to 10.78 + 2.62 and 6.92 + 0.71 >imol total
CO^ fixed.

40

"O

0

i_

0

>

O

o

0

CO

0

03

SZ

•*->

c

>

co

o

0

SZ

CL

1

o

41

the latter comprises a far greater percentage of the total fruit
volume.

14

Inward redistribution of C-assimilates initially fixed in green

peel of grapefruit could have occurred via entry into the extensive

vascular network in the flavedo, but photosynthates arising from

leaves presumbly would be unloading from phloem at or near the same

sites. Movement could also have proceeded slowly from cell-to-cell as

long as a favorable gradient of solutes existed. Sucrose and hexose

concentration at this stage of development are compatible with the

above interpretation. Another possibility is that of internal

diffusion and refixation of respiratory 1<4CC>2, favored by extensive

air spaces in albedo, and by the heavy cutinization of the flavedo.

14

Localization of C-assimilates 1 h after exposure of fruit to
14

CC>2 in the dark changed only within peel during the subsequent 6 and

24 h (Fig 3-4), indicating that radioactivity recovered in juice

tissues was the likely result of carboxylation reactions at the same

14

site. This also implies that ample C02 could diffuse from the

atmosphere into juice tissues despite the much higher C09

concentration reported inside the fruit by Eaks and Ludi (1960). The
14

percentages of C-assimilates in albedo increased over the 24 h

period after initial fixation (Fig. 3-4). The total recovered dpm did

not differ significantly between 1 and 6 h, however (data not shown).

Albedo appeared to have the lowest respiratory rate when individual

fruit tissues were measured (data not shown). The greater percentage
P 14 .

ot C-assimilates recovered in albedo relative to other fruit tissues
after 24 h may partially reflect a lesser loss of ^C-assimilates from
respiration and related activity in albedo.

Fig. 3 4. Distribution of ^C-assimilates within young grapefruit
('Foster' pink seedless clone j^l-26-39, 2— months old) after exposure
of recently-detached fruit to i4C02 (A) for 1 h, followed by (B)
ambient air for an additional 6 h or (C) 24 h in continuous dark.
Vertical bars denote SE of 4 replications. Fl=flavedo, Al=albedo,
Jt=juice tissues. Total mean dpm recovered at 1 h, 1+6 h, and 1+24 h
were 255,150 (+ 26,010), 140,474 (+ 24,873), and 255,717 (+ 7,813),
respectively.

43

14

A. 1h C09 (dark)

■a

0

i_

0

>

O

o

0

FI

60

20

Al

Jt

B. 1 h 14C02 & 6h

(dark)

CO

0

00

CO

0

0

l

O

60

FI

20

Al

Jt

1 A,

C. 1 h CO

& 24h
(dark)

60

20

44

Conclusions

14

Distribution of labeled assimilates after light and dark CO^
fixation by leaves and fruit was as follows:

14

1. Exported products of both light and dark CO^ fixation in leaves
were deposited primarily in juice sacs of fruit. More of the
assimilates were retained in leaves after dark CO^ fixation.

2. Fruit photosynthesis gave rise to assimilates which remained

primarily in flavedo and albedo 24 h later, thus probably contributing

little to growth of juice tissues.

14 14

3. C-assirailates from dark CC^ fixation in intact fruit were
localized primarily in juice tissues.

An assessment of the relative C contributed by each assimilate

source to fruit development at various stages of growth indicated that

leaf photosynthesis was most important when fruit were very young. As

fruit expanded, the proportion of C contributed by fruit CC^ fixation

either in light or in the dark increased. The potential significance
14

of dark CC^ assimilation to organic acid synthesis in fruit will be
addressed in subsequent work.

CHAPTER IV

ORGANIC ACIDS FROM LIGHT AND DARK CO FIXATION
BY LEAVES AND FRUIT OF LOW-ACID CITRUS MUTANTS

Introduction

Levels of organic acid in fruit can dramatically affect flavor
attributes such as sourness (Ulrich, 1970). The sugar to acid ratio is
also an important criterion for determining the maturity of citrus and
other fruits (Ulrich, 1970). Citric and malic acid, which are the main
organic acids in citrus fruit, are important to many aspects of plant
metabolism (Raven and Smith, 1976; Edwards and Huber, 1981; Lance and
Rustin, 1984; Bown, 1985). The involvements of organic acids in
photosynthesis, pH balance, and nitrogen assimilation are well known.
Fleshy fruits are a rich source of organic acids (Ulrich, 1970), but
the mechanism and control of synthesis and accumulation remain
unclear. Citrus fruit acidity is affected by many factors. Organic
acid content varies widely among fruit species (Thimann and Bonner,
1950; Ulrich, 1970), between fruit on the same tree (Sinclair, 1984),
and even at different locations within the same fruit (Ting, 1969). A
significantly higher ratio of citricrraalic acid occurs in sour types
of citrus fruit, with the reverse evident in less sour fruit. Citric
acid predominates in juice tissues but malic acid is greater in the
peel. The acid content in citrus fruit can also be affected by climate
(Reuther, 1973) and horticultural practices such as irrigation (Koo
and McCornack, 1965; Gilf illan et al. , 1976), fertilization (Koo and

45

46

McCornack, 1965), and arsenical sprays (Wilson, 1983).

Three hypotheses have been proposed to account for the possible
origin(s) of the mechanism of organic acids accumulation in citrus
fruit :

1. Organic acids may be produced in fruit by metabolism of
photosynthates transported from leaves (Varma and Ramakrishnan, 1956;
Ramakrishnan, 1971).

2. Organic acids may be transported unaltered, directly from leaves or
roots (as reviewed by Ulrich, 1970).

3. Organic acids may be formed in fruit by fixation of respiratory or
atmospheric C02 via phosphoenolpyruvate(PEP) carboxylase (Huffaker and
Wallace, 1959) and/or metabolism of carbohydrates produced during
photosynthesis in fruit.

Reciprocal grafting between fruit of sweet and sour lemons, and
their respective leafy branches, suggested that a primary mechanism
affecting organic acid accumulation was probably localized in fruit
rather than leaves (Erickson, 1957). Organic acid synthesis in the
fruit tissue presumably would occur through the combined operation of
the glycolytic pathway and TCA cycle (Varma and Ramakrishnan, 1956;
Ramakrishnan, 1971), utilizing carbohydrates which might arise either
from leaf or fruit photosynthesis (Bartholomew and Reed, 1948). No
direct evidence has been provided to support this hypothesis, although
it is favored by researchers studying grape (Ruf fner et_ al. , 1983).

Most organic acids found in fruit have been detected in exudates
from phloem. The type of organic acids varies with different plant
species. However, organic acids are only a minor component in phloem
exudates (Ziegler, 1975) and initially seem insufficient to explain

47

the large amount acid accumulated in citrus fruit.

The extent to which products of CC>2 fixation in fruit are
partitioned into organic acids is still unknown. Dark CC>2 fixation
could be an anaplerotic carboxylation reaction for amino acid and
lipid synthesis and may be related to a metabolic pH balance in cells
(Davies, 1973; Bidwell, 1983). Huffaker and Wallace (1959) suggested
that dark CC>2 fixation was related to acid accumulation in citrus
fruit and that PEP carboxylase was a key enzyme for C02 fixation. More
assimilates were recovered as organic acids after dark CC>2 fixation
than in light after photosynthesis by lemon fruit (Bean and Todd,
1960). However, the relationship between amounts of dark C02 fixation
and fruit acidity conflicted when different methods or parts of fruit
were used (Clark and Wallace, 1963; Bogin and Wallace, 1966a). The
actual source and pathway of organic acid synthesis in citrus fruit
are still unclear.

The objectives of this study were to clarify the role of light and
dark C02 fixation in organic acid accumulation by citrus fruit and to
quantify the amount of organic acid derived from leaves, peel and
juice tissues throughout fruit development. Two grapefruit clones were
also compared, which are known to differ from one another in that
levels of fruit acid are normal in one and low in the other (Hearn,
1986).

Materials and Methods

Normal- and low— acid grapefruit clones (seedless mutation #
1-26-39, and 1-26-51, respectively) were derived from 'Foster' pink
grapefruit (Citrus paradisi, Macf . ) by bud irradiation (Hearn, 1986).

48

Experimental trees were grown in Lake Wales, FL, and were 4— years old
on 'Savage' sour orange or sour orange rootstocks. Fruits and/or
branches with source leaves adjacent the fruit were randomly harvested
from 1 to 2 m above ground on the exterior of the canopy. Samples were
collected on May 7, June 25, July 27 and September 10, at about
40-day intervals, after 2, 3.5, 5 and 6.5 months' growth,
respectively. Several samples from the same trees were used for
different treatments and so that one tree constituted a replication.
Four replications were used for each experiment. Stems were
immediately immersed in water and transported to Gainesville for
experiments.

At each developmental stage, fruits or leaves adjacent to them

were enclosed in a cuvette and exposed to ^CO, (13.6 jiCi 1_1 ) for 1

h in light followed by ambient air for 24 h (12 h:12 h lightrdark

photoperiod). Cuvettes were completely sealed with a foil covering for

dark treatments. Levels of CO^ were monitored during experiments by

circulating air through an infrared gas analyzer (IRGA) (Model 865,

Beckman Instruments Inc., Fullerton, CA). Data were used to calculate

mean specific activities of 14CC>2 during periods when release of

respiratory C02 increased total levels. Temperature was maintained at

28°C and photosynthetic photon flux density at 800 to 1000
. -2 -1 T .

umol m s . Light was provided by a Lucalux lamp (Lu 400, General
Electric Co. Cleveland, OH).

Each experiment was terminated by separating tissues into flavedo
(pigmented portion of peel), albedo ( non-pigmented portion of peel)
and juice tissues. Samples were frozen in liquid nitrogen, extracted
in 80% (v/v) ethanol, and the radioactivity in each was quantified by

49

liquid scintillation (LKB , Gaithersburg, MD). The ethanol-soluble
fraction was measured by pipetting 0.1 ml of sample into 15 ml
scintillation cocktail (Scinti Verse II, Fisher Scientific Co., Fair
Lawn, N J ) . The ethanol— insoluble fraction was measured by suspending a
known portion of the sample in scintilation cocktail. Quench curves
were established for materials utilized, but were generally not needed
for conversion of cpm to dpm.

The ethanol-soluble fraction was further fractioned with

ion-exchange resin (Dowex l-x8) using a modified procedure of Atkins

and Canvin (1971). Organic acids were eluted from the anion resin with

30 ml 6 N formic acid and further analyzed by HPLC (3io-Rad, Richmond,

CA). Samples were first passed through a C18 Sep-Pak cartridge

(Water's Associates, Milford, MA) and 0.45 ja filters. A 20 ul sample

was then injected and separated using an ion exchange column (Aminex

HPX 87H, 300 X 7.8 mm) at 65°C. The mobile phase was 0.01N H SO at a

2 4

flow rate of 0.4 ml rain The ^C-citric and malic acid fractions
were collected (Gilson International Co., Middleton, WI) and measured
by liquid scintillation spectroscopy as mentioned above.

Results

Acid concentration decreased gradually during growth of grapefruit
with normal acid levels, but more rapidly in fruit of the low acid
clones (Fig. 4-1A). Early in development, juice tissues of the low
acid clone had a significantly higher acid content than did those of
its normal acid counterpart. The same result was observed the previous
season (not shown). Differences in acid levels were most evident in
juice tissues, but also in flavedo (pigmented portion of peel),

Fig. 4-1. The acid concentration (A) and total titratable acidity (B)
in juice tissues from fruit of normal and low acid grapefruit clones
in May, June, July and September, at about 2, 3.5, 5, and 6.5 months
of development. Vertical bars denote SE of 4 replications, and are
smaller than symbols where not pictured.

Titratable Acidity

51

Fruit age
(month)

5

6

7

52

followed by albedo (non-pigmented portion of peel) (data not shown).
The more rapid drop in acidity, however, brought levels from the low
acid clone to below those of the normal acid clone after June (Fig.
4-1A). At 3.5 months of development or after, total titratable acidity
of juice per fruit (Fig. 4-1B) was also significantly lower in the
clone known to have less acid at commercial harvest.

Further analysis by HPLC showed that citric and malic acids were
the primary organic acids in juice tissues of both grapefruit clones
(Table 4-1). Values are comparable to those previously reported for
grapefruit with normal levels of total acidity (Monselise and Galily,
1979). The ratio of citrate to malate was lowest early in development
and increased rapidly after June. In September, citric acid levels
were 14- and 9-fold greater than those of malic acid for fruit of
normal and low acid grapefruit clones, respectively.

Quantification of label in C-organic acids recovered from whole

fruit indicated that organic acid synthesis continued throughout

development (Fig. 4—2). About 1% or less of the labeled photosynthates

translocated to fruit from an adjacent leaf had been converted to

organic acids of juice tissues 24 h after initial 14C02 fixation (Fig.

4-2A). Values were greater when organic acids were expressed as
14

percentages of C-assimilates in juice tissues alone (Fig. 4-2B). In
both instances, the extent of organic acid formation from imported
leaf photosynthates dropped by 50% or more between the 2nd and 4th
months of growth. No significant differences were noted between normal
vs. low acid fruit.

In contrast to labeled photosynthates from adjacent leaves, those
derived from fruit photosynthesis were recovered to an increasing

53

Table 4-1. The ratio of citric to malic acid in juice tissues during
development of fruit on normal and low acid grapefruit clones.

Ratio of citrate/malate

(Citric acid/malic acid)

May June

July Sept

Normal Acid Mutant
Low Acid Mutant

10.6 + 1. lz 10.0 +0.7 13.8
9.4 + 0.7 9.2 + 0.6 9.4

Means of 6 replications +_ SE (6 fruits from 6 different trees) for
June, July, and September.

+ 1 +1

Fig. 4-2. Developmental changes in the percentage of 14C-assimilates
partitioned into organic aci<jl| of juice tissues 24 h after a 1 h
period of photosynthesis in 14CC>2 by an adjacent leaf (A, B), by an
intact, detached fruit (C, D) or lh of dark 14C02 fixation by an
intact fruit (E, F) from normal and Jow acid grapefruit clones.
Figure^on the left (A,C,E) denote C-organic acid percentages of
total C-assimilates translocated to, or retained in fruit. Those on
j^e right (B,D,F) show C-organic acids as percentages of total
C-assimilates translocated to, or retained in juice tissues.
Experiments were conducted using fruit approximately 2, 3.5, 5, and
6.5 months of growth. Note the differences in percentage units among
the figures. Vertical bars denote the SE of 4 replications, but are
present only where larger than symbols.

% of whole fruit dpm recovered as
4C-organic acids in juice tissues

55

1.6

0.8

1.6

0.8

40

20

Month AMJJAS AMJJAS

i i i i i i i i i 1 1 1 1 1

Fruit age 234567 234567

(month)

% of juice tissues dpm recovered as '"C-organic acids

56

extent as organic acids during fruit development (Fig. 4-2 C and D).
This was true even when only the ^C-assimilates in juice tissues were
considered (Fig. 4-2D). More than 50% of the fruit 14C-photosynthates
localized in juice tissues were organic acids at later stages of fruit
growth (September) (Fig. 4-2D). Differences between fruit of normal-

and low-acid lines were not significant.

14

A greater percentage of C-assimilates were partitioned into
organic acids after dark CC>2 fixation by fruit than after
photosynthesis by either leaves or fruit (note units in Fig. 4-2 E and
F). Values ranged from 40% to 60% and were highest in fruit about 3.5
months old. Percentages may have been affected by the extent of amino
acid synthesis from organic acid products of dark 14CC>2 fixation.
Again, fruit of the two grapefruit clones did not differ
significantly.

Figure 4-3 shows the amount of organic acids synthesized in juice
tissues from assimilates produced during 1 h of photosynthesis by a
single, adjacent leaf, by the intact fruit, or by dark C02 fixation in
the fruit. These data were used to estimate the extent of total,
overall organic acids arising directly or idirectly from the sources
described in Table 4-2. Equations and parameters employed were those
discussed in Chapter III (Table 3-2). Calculations considered
differences in overall input from leaves relative to changes in CC>2
levels during experiments, possible and reported numbers of leaves
involved, the portion of a 24 h day in which a given process would be
expected to occur. Results appeared that the major sources of organic
acid formation during early fruit growth were photosynthates
translocated from adjacent leaves and dark CC>2 fixation in fruit

Fig. 4-3. The jamol of organic acids contributed to juice tissues 24 h
after a 1 h period of C0„ fixation by either in an adjacent leaf in
the light (A), fruit in the light (B), or fruit in the dark (C).

Values were calculated from the 14C-organic acids formed after
fixation (Fig. 4-2), from expected dif f erence|^in the numbers of C
atoms labeled during photosynthesis vs. dark^ CC^ fixation, and from
changes in specific activity of atmospheric CO^ determined from
infrared gas analysis of total CO ^ levels during exposure. Vertical
bars denote the SE of 4 replications, which is shown only where larger
than the symbols used.

58

Fruit age 2 3 4 5 6 7

(month)

59

(Table 4-2). Later in fruit development, however, dark CO^ fixation by
fruit increased in importance and became one of the major sources of
organic acids. In contrast, fruit photosynthesis appears to play only
a minor role in organic acid synthesis.

Discussion

Decrease of organic acid concentration toward maturity has been
widely reported in citrus fruit (Ting and Vines, 1966; Erickson, 1968;
Sinclair, 1984). A comparison of Fig. 4-1A to B indicates that
decreases in acidity are due to fruit expansion, and that total
organic acid accumulation occurs continuously throughout fruit
development. It is unclear why low acid fruit accumulate organic acids
less rapidly, especially after the 5th month of fruit development.

Results confirmed that citric and malic acids accounted for most
of the organic acids in juice tissues of grapefruit (Monselise and
Galily, 1979), but the ratio of citrate to malate was much greater
throughout development. Differences between fruit of the two clones

were singnificant in September (Table 4-1).

14

The percentages of C-assimilates partitioned into organic acids
were significantly different between various sources (Fig. 4-2). This
may be associated with the function of different sources in fruit
growth (especially when compared with Chapter III). About 40% to 60 %
of the assimilates derived from dark C02 fixation by juice tissues
remained as organic acids 24 h later (Fig. 4-2). This is similar to
percentages reported by previous researchers (Bean and Todd, 1960;
Young and Biale, 1968).

The initial product of dark CO^ fixation is oxaloacetate (OAA) ,

60

Table 4-2. The contribution of various sources to organic acids in
juice tissues of normal and low acid grapefruit during fruit
development.

2

Time Source leaves Fruit

Light Dark Light Dark

(% of contribution to organic acids)

Normal acid

May

94.0

to

98.4

0.1

0.02

to

0.06

1.5

to

5.8

June

30.9

to

64.1

NDy

0.9

to

1.8

35.0

to

67.3

July

18.0

to

46.7

ND

2.5

to

3.9

50.8

to

78.2

Sept

6.6

to

22.0

ND

1.6

to

1.9

76.4

to

91.5

Low acid

May

93.8

to

98.4

0.1

0.03

to

0. 1

1.6

to

6.0

June

34.2

to

67.5

ND

0.4

to

0.8

32. 1

to

65.0

July

36.4

to

69.6

ND

0.8

to

1.7

29.6

to

62.0

Sept

12.4

to

36.2

ND

1.3

to

1.8

62.6

to

85.8

2 t tf

Values estimated from CO^ fixation by fruit represent minimal
estimates due to high internal CO^ levels and extensive refixation
of respiratory CO^*

y

not detected at later stages

61

which is converted immediately to malate (Latzko and Kelly, 1983).

Less than 8 minutes were required to turn over malate produced during

dark CC^ fixation in tomato fruit, possibly reflecting the amount

present in the cytoplasm (Farineau and Larval-Martin, 1977). Turnover

is 157 minutes, however, when malate is stored in vacuoles of root

cells (Ting and Dugger, 1966). In their experiments with tomato,

14

Farineau and Laval-Martin (1977) found that 50% of C-3-malate

infiltrated into tomato flesh in the dark was recovered in C, acids

6

(citrate and isocitrate). Malate can apparently turnover rapidly in

plant cells during anaplerotic dark CO^ fixation. In the present

study, a ratio of 1.3 for labeled citrate/malate was observed in juice

14

tissues after dark CO^ fixation by fruit in July. This is

considerably less than the ratio of total citrate to malate found in

juice tissues (from 9:1 to 14:1). A rapid and highly labeled

14

release after dark CO^ fixation has been reported by Splittstoesser

(1966). Therefore, the more extensive label in malate may be due to a

slower turnover of acid when stored in vacuoles, or a continuous

14

refixation of respiratory CC^ released. Together these two acids
accounted for about 90% of the of label in the anion fraction.

Although dark CC^ fixation appears to be a major source of organic
acids in grapefruit after 3 months' growth, leaf photosynthates still
play an important role by serving as substrates for synthesis of
phosphoenolpyruvate (PEP), precursor of the dark CO^ fixation
reaction. Dark CC^ fixation by leaves was shown not to be a
significant source of fruit organic acids.

The mechanism of biosynthesis and sources of organic acids in
citrus fruit have been debated for many years (Bartholomew and Reed,

62

1948; Varma and Ramakrishnan, 1956; Huf faker and Wallace, 1959;

Buslig, 1968; Ulrich, 1970; Ramakrishnan, 1971; Kubota and Akao,

1973a; Akao and Kubota, 1978b; Akao and Tsukahara, 1980b) and several

hypotheses have been suggested. Results of the present study indicate

that leaf photosynthates are the primary source of organic acids in

very young citrus fruit (Table 4-2) depite the small amount

partitioned into fruit acids from individual leaves (Fig. 4-2 and

4-3). Fruits receive photosynthates from numerous leaves rather than

one, and about 60 leaves are required to support normal growth of a

single grapefruit (Shamel and Pomeroy, 1934).

Calculations based on Fig. 4-3 (Table 4-2) indicated leaf

photosynthates were the predominant source of organic acids during

early fruit growth, but decreased rapidly as dark CC^ fixation become

a more important contributor of organic acids at later stages.

Calculated values for total organic acid synthesis after light
14 12

fixation of CC^ + CO^ by intact fruit represent minimal amounts.

14

Dilution of atmospheric CO^ by unlabeled, respiratory CC^ was
measured and accounted for, but not refixation of unlabeled C07
retained within tissues. The same holds true for both light and dark
CC>2 fixation by fruit.

Conclusions

Acid concentration decreased and total acidity increased
continously throughout development of fruit from both normal- and
low-acid grapefruit clones, but values were significantly less in
grapefruit from the low-acid clone after 5 or more months' growth. In
more mature fruit, the citrate to malate ratio was also significantly

63

less in the low- vs. high-acid fruit, 9:1 and 14:1, respectively.

Significant differences were not evident in amounts of
14„ , ,

C-assimilates partitioned into organic acids in fruit of normal- and

low-acid grapefruit clones, but may have resulted from the relatively

short initial exposure time (1 h) compared to the duration of fruit

development. About 90% of anion fraction of ^C-assimilates recovered
14

from dark CO^ fixation in juice tissues of 5-month-old fruit was
citrate and malate. The ratio of labeled citrate to raalate was far
less than that of total levels in juice tissues, possibly due to slow
turnover of raalate in vacuoles or continuous refixation of in

the dark.

Leaves were the primary source of assimilates for organic acid
formation early in fruit development. However, dark CO^ fixation by
fruit became a major, and perhaps predominant source of organic acids
later in development, despite of its relatively minor contribution to

total C.

CHAPTER V

EFFECTS OF ARSENATE ON ACIDITY AND LONG-TERM ASSIMILATE
PARTITIONING IN CALAMONDIN (Citrus madurensis Lour.)

Introduction

The acid level of citrus juice tissue decreases during
development, and is important to both quality of fruit and timing of
early harvests. A specific soluble solids/acid ratio is required
before fruit can be picked, and neither occur until acidity drops
below a level considered acceptable relative to total sugars. If this
occurs early in the season, a higher price may be obtained for fruit.
It has long been known that arsenate-based insecticides will
significantly decrease acidity of some maturing citrus fruit. Fruit on
sprayed trees can be harvested weeks earlier (Wilson, 1983).

It has also been shown that arsenate is the active agent in this
decrease of fruit acidity (reviewed by Wilson, 1983; Rice et al. ,
1985). Initially, it was thought that reactions affecting acid
accumulation might be located within the citrus fruit itself
(Erickson, 1957). However, natural background levels of arsenate were
unaffected in juice sacs at any time after trees were sprayed. This
result suggested the possibility that the primary effects of arsenate
spray on fruit acidity might be elicited within the leaves (Rice et
al. , 1985). Other studies, however, indicated that the arsenate
concentration in peel was higher after arsenate treatment (Gilfillan
et al. , 1976). The possible effects of a slight rise in arsenate

64

65

concentration in fruit peel cannot be excluded.

It has not been determined which site of arsenate action results
in decreased acidity of citrus fruit, but the question has generated
considerable interest over the years. Vines and Oberbacher (1965)
suggested that the action of arsenate as an uncoupler of
phosphorylation might be associated with the decrease of acidity.
Oxidation of pyruvate and sk-ketoglutarate , the intermediates of
respiration, are also influenced by arsenate (Beevers et al. , 1966),
while aconitase, also a key enzyme in acid metabolism, is unaffected
(Dickman, 1961). Arsenate must be present at a given site to have
these effects, however, and at concentrations many times greater than
the non- or rarely-detectable amounts in fruit. The possibility of
arsenate involvement in metabolism mentioned above is therefore
unlikely in citrus juice tissues.

Arsenate has been found to increase respiration of grapefruit
leaves and this in turn may be associated with the decreased acidity
(Miller et al. , 1933). Experiments with isolated pea chloroplasts also
indicated that arsenite affected light modulation and photosynthetic
induction by interferring with light activation of enzymes in the
reductive pentosephosphate cycle (Marques and Anderson, 1986). An
additional possibility is arsenate competition with Pi and its
influence on the triose P carrier of the chloroplast envelope. This
could affect both partitioning and export of photosynthatic products.
The effects of arsenate on actively growing leaves are evident
immediately in fruit and may persist for about 18 months (Miller j2t_
al. , 1933). This is not inconsistent with leaf effects on fruit,
because photosynthetic products move primarily from leaves to nearby

66

fruit. The influence of arsenate on decreased fruit acidity is not
broadly systemic, however, because only fruit from the sprayed portion
of trees were found to have less acid juice (Miller et al. , 1933).

The action site of arsenate and its relation with acidity reduction
therefore remains unclear.

Buslig et al. (1971) reported that calamondin trees provide
excellent material for screening the acidity reduction potential of
various chemicals. These trees will also flower and fruit when very
small, so allow greenhouse and laboratory studies to be conducted on
mature, bearing citrus trees. The purposes of experiments described
here are 1) to compare the effects of arsenate on fruit acidity when
applied to individual fruit versus whole plants, and 2) to compare
assimilate partitioning in arsenated and non-arsenated calamondin
trees. In this way, one can determine to what extent leaf and fruit
sources of organic acid are influenced by arsenate treatment.

Materials and Methods

Calamondin (Citrus madurensis. Lour.) fruit, were used in
these studies not only because trees are small, but also because they
flower and fruit continuously (Hodgson, 1967). Plants were grown in 15
cm plastic containers (about 2.25 liters) in a greenhouse, and were
about 2-years old and 80 cm tall when used. For the first group of
experiments, plants were sprayed with arsenate (1.92 g 1 ^ ) when
fruits were one month old, as described by Deszyck and Ting (1958).
Four plants were used for each treatment, and 4 fruits were harvested
for measurement of juice acidity when they were 3- and 6-months old.
The effect of arsenate on whole plants, was compared to treatment of

67

fruit alone by painting 2-month-old fruit with the same concentration

of arsenate as applied to the whole tree. Five fruits were treated on

each of 3 plants, and another 3 trees were used for full-spray

treatments. Fruit acidity was determined by titration of juice from

fruit harvested 3 months after arsenation.

The second group of experiments compared assimilation,

partitioning, and allocation by leaves of fruit of arsenated and

14

non-arsenated trees. Leaves were exposed to CO^ in the light and
14

fruit CC>2 fixation was studied in both light and dark. Fruits were

about 3 months old and the fully expanded leaves adjacent to them were

14

selected as source leaves for CO,, assimilation. Either fruits or

14

adjacent leaves were enclosed in a cuvette and exposed to C02 (16.0

juCi 1 for 1 h followed by ambient air for 7 days. During

treatments, temperature was maintained at 28°C and photosynthetic

-2 -1

photon flux density at 800 to 1000 /nmol m s . Light was provided by

a Lucalox lamp (General Electric Co., Cleveland, OH). Cuvettes were

14

completely sealed with foil for C02 exposure in the dark. Plants

were transferred to a greenhouse the day following treatment.

14

Seven days after this exposure to C02 fixation, source leaves,
other adjacent leaves, and fruit were harvested. Fruit were further
dissected into peel and juice tissues. Samples were boiled in 80%

(v/v) ethanol and homogenized using a Polytron (Brinkraann Instruments,
Westbury, N.Y.). Ethanol-soluble and -insoluble fractions were
separated by filtration in a Buchner funnel. Radioactivity in each
sample was quantified by liquid scintillation (LKB, Gaithersburg, MD).
The ethanol-soluble fraction was measured by pipetting 0.1 ml sample
into 15 ml scintillation cocktail (Scinti Verse II, Fisher Scientific

68

Co., Fair Lawn, NJ). The ethanol-insoluble fraction was measured by
suspending the sample in scintillation cocktail. Ethanol-soluble
assimilates were further fractioned with ion-exchange resin (Dowex
1-X8) (Sigma, St. Louis, MO) by a modification of the procedure of
Atkins and Canvin (1971). The organic acid fraction was eluted from
the anionic resin with 30 ml 6N formic acid.

Results and Discussion

Decreases of acidity by arsenate treatment were significant in one
but not two experiments (Table 5-1). The time of arsenate application
might account for the different results. Arsenate treatments did not
decrease mean acid levels in calamondin juice tissues when the fruit
were arsenated in January and March (Table 5-1, Exp. I). Response
after both treatments were also less dramatic than those previously
reported by Buslig et al. (1971) who recommended calamondin as a plant
for screening chemicals for acid reduction potential. Decreased
acidity of citrus fruit appears to be a relatively consistent response
to whole-tree arsenate treatment, however, and has been identified in
grapefruit, orange, satsuma mandarin and calamondin (Buslig et al. ,
1971; Akao and Kubota, 1978a; Wilson, 1983).

Results here show that arsenate in June significantly decreased
the acidity of calamondin fruit even if only the fruits were treated
(Table 5-1, Exp. II). Differences between fruit and whole tree
treatments were not significant. Therefore, the decrease of acidity in
citrus fruit can potentially be brought about by the action of
arsenate in the fruit alone. Whole-tree sprays are typically applied
shortly after flowering, however, when fruit are so small that

69

Table 5-1. Arsenate treatments and acidity (meq g * fresh wt) of
calamondin fruit.

Exp. I.z

Arsenated

Non-arsenated

(meq g ^ fresh wt)

Young fruit

0.94 + 0.04

1.04 + 0.09

Mature fruit

0.91 + 0.07

0.94 + 0.07

Exp. II. y

Arsenated

Non-arsenated

Fruit

only

Whole plant

(meq g 1

fresh wt)

1.09 +

0.02

1.07 + 0.06

1.17 + 0.02

Trees were arsenated when fruit were about 1-month old. Samples were
taken on May 19, 1987, when young fruit were about 3-months old and
mature fruit about 6 months old. Each value is the mean of
measurements from 4 trees, with 4 fruit samples from each.

y

Trees were arsenated on June 3, 1987, when fruit were about 2 months
old, and harvested on Aug. 12, 1987, when fruit were about 5 months
old. Each value is the mean of measurements from 3 trees, with 3
fruit sampled from each.

70

arsenate interception would presumably be very small. Also arsenate

residue in grapefruit juice sacs is negligible (Rice et al., 1985) and

would therefore appear unlikely to be involved in the decrease of

fruit acidity. Still, in some instances arsenate has been found in

peel of sprayed trees (Gilfillan et al., 1976). The peel may therefore

have some effects on acid accumulation in juice sacs, at least in

calamondin fruit where the outer tissues (especially albedo) are

thinner than those of most other citrus fruit.

14

The relative amounts of C-assimilates arising from different

plant parts and later recovered in calamondin fruit (Table 5-2) were

similar to those found previously in grapefruit (Chapter IV).

Photosynthesis in single leaves of either arsenated or non-arsenated

12 14

trees were the largest source of total C- plus C-assimilates in

calamondin fruit, followed by photosynthesis in fruit. Data in Table

5-2 also show that arsenate treatment of calamondin trees had no

significant effect on amounts of leaf or fruit photosynthates

recovered from fruit 1 week after assimilation. In contrast,

14

significantly more C-assirailates were localized in fruit of

14

arsenated trees 1 week after CC^ fixation by fruit in the dark. In
other experiments, it appeared that dark CC^ fixation via
phosphoenolpyruvate carboxylase was a major source of organic acids
but the control point of organic acid accumulation seemed more likely
to be associated with citrate synthase-aconitase (Chapter IV and VI).
At the same time, the sum total of assimilates derived from dark C0?
fixation in fruit is extremely small when compared to amounts arising
from photosynthesis in green fruit tissues or a single leaf (Table

5-23).

71

Table 5-2. Total dpm and umol recovered from 3-month old calamondin
fruit 7 days after a 1 h exposure of either leaves or fruit to 1 CO
in light or dark.

A. Total dpm

recovered from fruit

Single leaf

Fruit

Fruit

in light

in light

in dark

(dpm x 107)

(dpm x 107)

(dpm x 10^)

Arsenate

7.018 + 1.196

2.838 + 0.394

6.123 + 1.562

Non-arsenated

5.350 + 1.080

3.237 + 0.252

3.839 + 0.422

12 14

B. Total exogenous C+ C assimilated as CO 2 and

subsequently

recovered

in fruit

Single leaf

Fruit

Fruit

in light

in light

in dark

(pmol)

(pmol)

( pmo 1 )

Arsenate

4.509 + 0.768

1.666 + 0.231

0.058 + 0.015

Non-arsenated

3.437 + 0.694

1.901 + 0. 148

0.036 + 0.004

Values are calculaj^d on the basis of measured changes in specific
activity of CC>2 + CO^ during the assimilation period, and expected
lumbers of C atoms labeled in products of photosynthesis vs. dark
CC>2 fixation.

72

When photosynthate distribution was compared 7 days after leaf
14

CC>2 fixation in the light, a significantly smaller percentage of the
14

total C-assimilates was recovered in the insoluble fraction of

leaves from arsenated trees, and more of the 1ZfC-photosynthates were

translocated to fruit (Table 5-3). More of the leaf-derived
14

C-photosynthates were localized in the peel of arsenate treated

plants vs. non-arsenated plants. A larger portion of these labeled

assimilates was also present in the ethanol-insoluble fraction

(predominantly starch and structural constituents) of both juice

tissues and peel of fruit from arsenated trees. The amount of
14

C-photosynthates from leaves recovered in juice tissues were not
significantly different between these two treatments. Differences in

distribution might be due to either altered photosynthate partitioning

14

and translocation from leaves, or greater rates of respiratory CO^
loss from leaves of arsenated plants. Both may be involved. Higher
respiratory rates have been found in leaves of arsenate-treated
grapefruit (Miller et al. , 1933).

14

Partitioning and distribution of C-photosynthates 7 days after

fruit CC>2 fixation in light were similar in fruit from arsenated and

non-arsenated trees (Table 5-3). Peel retained a greater portion of
14

C-assimilates than it had acquired after either photosynthesis by

leaves or dark CC^ fixation in fruit. A larger fraction of total

labeled assimilates was also recovered as ethanol-insoluble compounds

after fruit vs. leaf photosynthesis. About 23% of the fruit
14

C-photosynthates were recovered in juice tissues of fruit from both

arsenated and non-arsenated trees. Akao and Tsukahara (1980a) also

14

found about 25% of the C-photosynthates were in juice tissues 5 days

73

Table 5-3. Assimilate distribution among tissues of arsenated and
non-arsenated calaraon^n trees 7 days after a 1 h exposure of either
leaves or fruits to CO in light or dark.

Treatments Source leaf and Peel Juice tissues

adjacent leaves

Single leaf (Light)
Arsenated S^ 19.13

I 4.91

Non-arsenated S 23.83

I 10.83

(% of total recovered ^C)

+ 1.69 11.71 + 0.81
+ 0.32 17.44 + 2.36
+ 1.41 9.99 + 1. 18
+ 2.08 11.79 + 2.27

32.72 + 2.39
14.09 + 0.79
34.58 + 2.97
8.97 + 0.84

Fruit (Light)
Arsenated S
I

Non-arsenated S
I

33.44 + 1.52
42.23 + 6.68
32.69 + 1.50
43.35 + 2.11

18.76 + 4.54
5.35 + 1.34
19.75 + 1.41
3.97 + 0.71

Fruit (Dark)
Arsenated S
I

Non-arsenated S
I

13.98 + 4.52
12.54 + 5.75
12.38 + 0.79
8.59 + 1.50

68.29 + 9.65
4.16 + 0.44
72. 18 + 2.95
3.46 + 0.42

Fruit were about 3 months old.

y

S= ethanol-soluble fraction, including mono- and dissacharides ,
organic acids, amino acids, essential oils, and other undetermined
constituents.

1= ethanol-insoluble fraction, including starch, cell wall or
structural components such as cellulose, lignin, proteins, pectic
substances and hemicellulose (Sinclair, 1984).

74

14

after satsuma mandarin fruit were exposed to CO^ in light. However,

previous experiments with grapefruit showed that less than 4% of the
14

C-assimilates from fruit photosynthesis were present in juice

tissues after comparable periods of time (Chap. III). Albedo of

calamondin and satsuma madarin peels is considerably thinner than that

of grapefruit. Peel thickness therefore may be important in the extent

to which photosynthesis contributes to juice tissues.

Assimilates recovered 7 days after dark fixation in juice

tissues was partitioned similarly in fruit from arsenated and

non-arsenated trees (Table 5-3). In both, a much greater percentage of
14

C-assirailates were localized in juice tissues than had been after
14

light CO^ fixation, although the total amount was far less. A higher

ratio of ethanol-soluble to ethanol-insoluble assimilates in juice

tissues suggested a different fate for products of dark vs. light CC^
14

fixation. The C-assimilates in the insoluble fraction of peel

extracts probably arose either through photosynthetic refixation of
14

respired CC^, or synthesis of structural components from amino acids

14

produced after dark CC^ fixation.

Assimilate partitioning into organic acids of fruits from
arsenated and non-arsenated trees was similar after either light or
dark CO^ fixation in fruit but greater amounts of ^C-photosynthates
from leaves were recovered in the anion fraction (predominantly
organic acids) of fruit from arsenated trees (Table 5-4). Also, the
total amounts of labeled and unlabeled photosynthates converted to
organic acids of fruit was estimated from total CO^ fixation, and
found to be larger in arsenated than non-arsenated trees. The result
might initially appear to conflict with the role of arsenate in

75

Table 5-4. Assimilate partitioning into the anion fraction
(predominantly organic acids) of juice tissues of arsenated and
non-arsenated calamon^n trees 7 days after a 1 h exposure of either
leaves or fruits2 to C02 in light or dark.

dpra recovered in
Treatments anion fraction
of juice tissues
(dpm 103 g"1)

Anion fraction
expressed as
total C fixed
(jimol g *)

7o of anion
fraction recovered
in juice tissues
(%)

Leaf (Light)

Arsenated

Non-arsenated

Fruit (Light)

Arsenated

Non-arsenated

Fruit (Dark)

Arsenated

Non-arsenated

460.2 + 50.3
243.0 + 81.4

66.0 + 13.7
73.5 + 14.0

3.8 + 1.1

4.1 + 0.4

0.296 + 0.032
0.156 + 0.052

0.039 + 0.008
0.043 + 0.008

0.004 + 0.001
0.004 + 0.001

33.30 + 0.54
33.16 + 2.02

32.84 + 3.92
34.32 + 1.03

63.77 + 2.20
60.13 + 5.99

z

Fruit were 3-months old

76

14

decreasing the acidity of citrus fruit, but when C-phtosynthates in
the anion fraction of juice tissues are expressed as a percentage of
the amount translocated into these tissues, arsenated and
non-arsenated fruit do not differ (Table 5-4). Arsenated trees
reportedly show no detectable increases in arsenate of juice tissues
(Gilfillan et _al. , 1976; Rice et al . , 1985). Therefore, greater
photosynthate export from leaves of arsenated trees could be
responsible for results observed here.

Data in table 5-4 also indicate that organic acids in citrus fruit

may arise from both light and dark CC^ fixation by fruit as well as

photosynthesis in leaves. The largest contribution to organic acid

synthesis was from leaf photosynthesis, followed by fruit CC^ fixation

14

in light. More than half of the C-assimilates resulting from dark
C02 fixation in fruit were partitioned into the anion fraction, but
resulted in only a very small total amount of organic acids in
calaraondin fruit. This differs from previous findings for grapefruit,
which showed that dark CO^ fixation was a major source of organic
acids in fruit (Chap. IV). The different fruit size and peel
characteristics may affect the amount of respiratory CO^ retained
inside juice tissues, which in turn could influence dark CC^ fixation
and its contribution to organic acids.

Conclusions

The effects of arsenate spray on calamondin fruit acidity were
variable, but in June experiments, application of arsenate to whole
trees or attached, individual fruit decreased acidity of juice
tissues. Arsenical residue in leaves is therefore not necessarily

77

involved in the typical response to whole-tree sprays. However,
effects of arsenate on leaves and/or fruit peel are likely to be
important under field conditions because arsenical residues in juice
tissues do not differ significantly after arsenate spray (Gilfillan jrt
al. , 1976; Rice et al. , 1985).

Leaf metabolism appeared to be altered by whole-tree arsenate

treatments in the present study, but not that of fruits. Partitioning

14 14

of C-assimilates 7 days after light and dark CO^ fixation by

leaves and fruits of calamondin trees showed that greater percentages

of leaf photosynthates were translocated to fruit of arsenated trees.

A larger portion of the total assimilates were thus found in the

organic acid fraction of fruit. Less total photosynthate was recovered

in arsenated trees, however, possibly due to elevated respiratory

rates of arsenate-treated leaves reported by Miller et al. (1933).

14

Percentages of C-assirailates partitioned into organic acids did not

14

differ significantly if derived from CO^ fixation by fruit.

The contribution of fruit photosynthesis to growth of calamondin
juice tissues appears to be greater than that of grapefruit (23% vs
less than 4%) and may be related to differences in peel thickness.

CHAPTER VI

ENZYMES OF ORGANIC ACID SYNTHESIS AND METABOLISM DURING
DEVELOPMENT OF NORMAL AND LOW ACID GRAPEFRUIT MUTANTS

Introduction

The organic acid content of fruit is a critical factor in flavor
and overall quality. Seasonal and developmental changes in organic
acids, as well as associated synthesis and degradation, have been
widely studied in many fruit crops (Thimann and Bonner, 1950; Ulrich,
1970; Sinclair, 1984). Several hypotheses have been proposed to
account for acid biosynthesis, based particularly on data from grape
(Ulrich, 1970; Ruffner et al. , 1983) and citrus (Bartholomew and Reed,
1948; Varma and Ramakrishnan , 1956; Huffaker and Wallace, 1959;

Buslig, 1971; Akao and Tsukahara, 1980b). Organic acids in grape
vines, for example, can be transported from leaves to fruit (Ulrich
1970), although recent evidence indicate this is not likely the main
source of fruit acids (reviewed by Ruffner, 1982). In citrus,
reciprocal grafts between sour and sweet lemons suggested that the
primary mechanism of organic acid synthesis is probably localized in
the fruit itself (Erickson, 1957).

Enzymes associated with organic acid accumulation in citrus fruit
have been investigated and reviewed (Ericson, 1968; Kefford and
Chandler, 1970; Vandercook, 1977; Bruemmer et al, 1977; Sinclair,
1984). Dark CO^ fixation via phosphoenolpyruvate carboxylase (PEPc)
was suggested to be associated with organic acid synthesis in citrus

78

79

fruit (Huf faker and Wallace 1959). Activity of PEPc is higher during
the period of rapid organic acid accumulation in several citrus fruits
(Clark and Wallace, 1963). However, the same and other studies showed
that the PEPc acitivities among different citrus cultivars did not
correlate with respective fruit acid levels (Clark and Wallace, 1963;
Bogin and Wallace, 1966). Phosphoenolpyruvate carboxylase activity was
highest in citrus fruit with lowest acidity. The relationship between
acid accumulation and the sequence of reactions from CC^ fixation to
citrate synthesis was thus found to be more complicated than initial

hypothesis had proposed (Bogin and Wallace, 1966).

14

Dark CO^ fixation in lemon fruit, however, resulted in extensive
labeling of organic acids (Young and Biale, 1968). Similar results
have also been reported for a wide range of citrus fruit (Vu et al. ,
1985; Chapter IV). Fruit assimilation of CO^ in the dark was found to
be a major source of organic acids in developing grapefruit (Chapter
IV). Dark CC^ fixation also occurs in flavedo and albedo, but does not
result in as extensive an accumulation of organic acids (Bean and
Todd, 1960).

Citrate synthase and aconitase in the tricarboxylic acid (TCA)
cycle are two enzymes directly associated with synthesis and
degradation of citric acid. Both enzymes have been reported in leaves
and fruit of several citrus varieties (Ramakrishnan and Varma, 1959;
Srere and Senkin, 1966; Buslig, 1971; Hirai and Ueno, 1977).
Ramakrishnan and Varma (1959) found that in lemon fruit, activity of
citrate synthase increased and that of the degradative enzymes,
aconitase and isocitrate dehydrogenase, decreased during the period
when the acid content rose from 0.9% to 4.0%. Bruemmer et al. (1977)

80

reviewed the enzyme systems controlling citric acid synthesis in
citrus and suggested that citrate synthase was a probable regulatory
enzyme for this process as well as for organic acid degradation in
citrus fruit. Buslig (1971) suggested that an asynchrony between
citrate synthesis and degrading enzymes, such as isocitrate
dehydrogenase, might occur during citrate accumulation. Bogin and
Wallace (1967) proposed that citramalate, which inhibits aconitase,
may contribute to the acid accumulation in citrus fruits. However,
there is yet no direct evidence demonstrating that citrate synthase
and aconitase are actually involved in acid accumulation by citrus
fruit.

Excellent material for comparison of acid metabolism is provided
by plants which are genetically identical, or nearly so, except for
attributes affecting fruit acidity. Such clones have been developed by
bud irradiation of 'Foster' pink grapefruit (Hearn, 1986). Two of the
seedless mutations which arose apparently differ from one another in
levels of fruit acid; one normal, and the other low.

The first objective of this study was to characterize
developmental changes in activities of enzymes potentially associated
with acid accumulation in grapefruit tissues. The second was to
compare such data from the grapefruit mutants which had either normal
or low fruit acidity. Combined results should help clarify mechanisms
affecting the acid metabolism in citrus fruit.

81

Materials and Methods

Plant Material

Grapefruit mutants with normal and low levels of fruit acid
(seedless clone # 1-26-39 and 1-26-51, respectively) were derived from
'Foster' pink grapefruit (Citrus paradisi, Macf.) by bud irradiation
(Hearn, 1986). All material was propagated vegetatively. Experimental
trees were 4-year old on sour orange rootstock in Lake Wales, FL. The
two seedless mutants appear to differ from one another in their fruit
acidity. Isozyme banding analysis of several enzymes and other data
showed no significant differences in characteristics tested between
the two clones (Yen, unpublished data, 1985).

Sampling

Fruits were randomly harvested from 1 to 2 m above ground on the
canopy exterior on May 7, June 25, July 27, and September 10
(approximately 40-day intervals), after 2, 3.5, 5, and 6.5 months'
growth, respectively. One fruit was harvested from each tree and 4
trees were used as 4 replications. Fruit were dissected into flavedo,
albedo and juice tissues, frozen immediately with liquid nitrogen, and
stored at -80°C until use.

Acid Levels

Acidity was determined by titration. Five g of each tissue were
homogenized with a Polytron (Brinkmann Instruments, Westbury, N.Y.) in
50 ml of deionized water. A 5 ml aliquot was again diluted 5:45 (v/v)
and titrated to an endpoint of pH 8.2 (using phenolphthalein
indicator) with 0.01 N NaOH at 25°C.

82

Enzyme Assays

Samples for enzyme assays consisted of either 1 g of flavedo
(outer, pigmented peel), 1 g of albedo (inner, non-pigmented peel), or
4 g of juice tissues. Each was homogenized at 0 to 4°C using Polytron
(Brinkmann Instruments, Westbury, N.Y.) with 10 ml of 0.2 M Tris
buffer (pH 8.2), 10 mM isoascorbate, and 0.1% Triton x-100 as
described by Hirai and Ueno (1977). The homogenate was filtered
through 6 layers of cheese cloth and centrifuged at 1000 x g for 10
min. Supernatant was used to assay protein levels and aconitase
activity, while other assays were conducted after removal of sugars,
acids, and other low molecular weight compounds (Hirai and Ueno 1977).
Extract was passed through a 10 g, Sephadex G-25-150 (Sigma, St Louis,
M0.) column (15 X 2.2 cm) previously equilibrated with 0.2 M Tris
buffer (pH 8.2) (Hatch and Oliver, 1978). The entire procedure was
conducted at 0-4 °C.

Protein was quantified according to Bradford (1976), using bovine
serum albumin standards.

Enzymes were assayed spectrophotometricaly at about 25°C.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity was determined

by measuring the decrease in NADH (A./n ) during a coupled reaction

340nm ° r

with malate dehydrogenase (Lane j^t _al. , 1969). The assay medium
contained 40 mM Tris buffer (pH 8.5), 10 mM KHCO^, 2 mM MgC^, 2mM
PEP, 0.5 mM glutathione, 0.06 mM NADH, and extract, in a final volume
of 2.5 ml. The reaction was initiated by addition of extract. Extracts
of grapefruit tissue had high enough activity of malate dehydrogenase
(MDH), that addition of exogenous enzyme was not necessary.

Malate dehydrogenase (EC 1.1.1.37) was assayed as described by

83

Asahi and Nishiraura (1973) in a reaction mixture of 75 mM phosphate
buffer (pH 7.0), 0.1 mM NADH, 0.75 mM oxaloacetic acid (OAA) and
extract in a final volume of 3 ml. The reaction was initiated by
addition of extract and quantified by spectrophotometric measurement
of decreases in NADH.

Citrate synthase (EC 4. 1.3.7) activity was determined according to
Srere (1969), by following production of mercaptide (A^^nm^ , formed
during the reaction of CoASH with Ellman's reagent,

5, 5 '-dithiobis-(2-nitrobenzoic acid) (DTNB). The assay medium
contained 40 mM Tris buffer (pH 9.0), 40 ;uM DTNB, 80 juM Acetyl CoA, 80
pM oxaloacetic acid, and extract in a final volume of 2.5 ml.

Aconitase (EC 4. 2. 1.3) was assayed by measuring the disappearance
of cis-aconitate absorption at 240 nm (Hirai and Ueno, 1977). The
reaction medium contained 40 mM Tris buffer (pH 7.5), 100 mM NaCl, 0.1
mM cis-aconitate, and tissue extract in a final volume of 2.5 ml. The
reaction was initiated by addition of extract.

Results

Acidity decreased gradually during development of all fruit
tissues from both the normal- and low-acid grapefruit clones (Fig.
6-1). This was due primarily to fruit expansion, because total acid
per fruit increased (data not shown). General changes in titratable
acidity of juice tissues were similar to those of other grapefruit
(Ting and Vines, 1966). Juice tissue acidity in the present study was
consistently greater than that of flavedo or albedo. Acid levels were
also significantly greater in all tissues of very young fruit from the
low-acid clone when compared to those of its counterpart known to have

Fig. 6-1. Acid levels during development of flavedo (outer, pigmented
peel) (A), albedo (inner, non-pigmented peel) (B), and juice tissues
(C) of fruit from grapefruit with normal acid levels ('Foster' pink,
seedless clone # 1-26-39), or low acid levels ('Foster' pink, seedless
clone # 1-26-51). Vertical bars denote the SE of 4 replications and
are not pictured where smaller than symbols used.

Titratable acidity (meq . g fresh wt)

85

Fruit age 2 3 4 5 6 7

(month)

86

normal acidity at harvest. However, mean titratible acidity of juice
tissues from the low-acid clone dropped rapidly between the 2nd and
4th months of fruit growth (May and June), so that by the 5th month
(July), acid levels were significantly less than those of fruit from
clones with normal acid levels.

Protein content decreased in flavedo, albedo, and juice tissues
throughout fruit development (Fig. 6-2). Differences were seldom
significant between grapefruit with normal acid levels when compared
to those of its low acid counterpart. Protein content was maximal in
the youngest fruit sampled (May, 2 mo) and was greatest in juice
tissues and flavedo. Levels in all tissues dropped during subsequent
fruit development, but did so most rapidly in expanding juice tissues.

Decreased protein concentration has also been reported during
enlargement (stage II) of 'Valencia' orange fruit, but was apparently
accompanied by an increase in soluble nitrogen (Bain, 1958).
Ramakrishnan (1971) suggested that the amino acids derived from
protein breakdown during this stage could be utilized as respiratory
substrates. Ribulose biphosphate carboxylase is not the primary
protein in citrus peel (Goldschmidt, 1986), but may acount for the
higher protein content observed in flavedo. Degradation of RuBP
carboxylase subunits does occur during final maturation of citrus peel
(Goldschmidt, 1986).

All enzyme activities (Fig 6-3 to 6-6) are expressed per g fresh
weight so that they can be more readily compared to organic acid
accumulations per g fresh weight.

Greatest activities of PEPc per g fresh weight were recorded for
extracts from the youngest juice tissues, which were also at their

Fig. 6-2. Protein content during development of flavedo (outer,
pigmented peel) (A), albedo (inner, non-pigmented peel) (B), and juice
tissues (C) of fruit from grapefruit with normal acid levels ('Foster'
pink, seedless clone // 1-26-39), or low acid levels ('Foster' pink,
seedless clone # 1-26-51). Vertical bars denote the SE of 4
replications and are not pictured where smaller than symbols used.

Protein (mg.g fresh wt)

88

Fruit age 2 3 4 5 6 7

(month)

Fig. 6-3. Phosphoenolpyruvate carboxylase (PEPc) activity during
development of flavedo (outer, pigmented peel) (A), albedo (inner,
non-pigmented peel) (B), and juice tissues (C) of fruit from
grapefruit with normal acid levels ('Foster' pink, seedless clone #
1-26-39), or low acid levels ('Foster' pink, seedless clone #
1-26-51). Vertical bars denote the SE of 4 replications and are not
pictured where smaller than symbols used.

90

Fruitage 2 3 4 5 6 7

(month)

91

most acid stage of development (Fig. 6-3). Activity dropped rapidly
during subsequent fruit growth, however, to levels less than those
from other tissues of the same fruit. Phosphoenolpyruvate carboxylase
from flavedo and albedo was generally more active early in development
and decreased gradually. Differences in PEPc activities were not
significant between extracts of fruit from clones with normal vs. low
acid levels. The amount and trend of PEPc activity during fruit
development was similar to that reported for soybean fruits and pea
pods, which reportedly reassimilate substantial respiratory CO^
(Quebedeaux and Chollett, 1975; Atkins £t jal_. , 1977). However, the
activity of PEPc from individual tissues was apparently lower than
those of flowers and leaves of 'Valencia' orange (Vu et al. , 1985).

Overall, trends in MDH activities per gram fresh weight were very
similar to those observed for PEPc (Fig. 6-4). Again, differences were
not significant between activities in extracts from fruit of clones
with normal vs. low acid levels. Developmental changes in activities
of citrate synthase in extracts of fruit tissues differed from those
of PEPc and MDH, primarily in the extent to which activity of flavedo
extracts was elevated between the 2nd and 6th months of growth (Fig.
6-5). In albedo, citrate synthase activities per g fresh weight
changed less during development, although, there was a slight decrease
from July to September. In contrast, citrate synthase in juice tissues
was similar to PEPc and MDH in that it was most active in extracts of
young fruit , when the acidity was greatest. Activities of citrate
synthase from grapefruit tissues were comparable to those previously
reported for lemons and oranges (Srere, 1966) and not significantly
different for normal and low acid grapefruit.

Fig. 6-4. Malate dehydrogenase (MDH) activity during development of
flavedo (outer, pigmented peel) (A), albedo (inner, non-pigmented
peel) (B), and juice tissues (C) of fruit from grapefruit with normal
acid levels ('Foster' pink, seedless clone // 1-26-39), or low acid
levels ('Foster' pink, seedless clone # 1-26-51). Vertical bars denote
the SE of 4 replications and are not pictured where smaller than
symbols used.

MDH activity (amol.g fresh wt . min

93

Fig. 6-5. Citrate synthase (CS) activity during development of flavedo
(outer, pigmented peel) (A), albedo (inner, non-pigmented peel) (B),
and juice tissues (C) of fruit from grapefruit with normal acid levels
('Foster' pink, seedless clone # 1-26-39), or low acid levels
('Foster' pink, seedless clone # 1-26-51). Vertical bars denote the SE
of 4 replications and are not pictured where smaller than symbols
used.

95

Fruit age 2 3 4 5 6 7

(month)

Fig. 6-6. Aconitase (AC) activity during development of flavedo
(outer, pigmented peel) (A), albedo (inner, non-pigmented peel) (B),
and juice tissues (C) of fruit from grapefruit with normal acid levels
('Foster' pink, seedless clone # 1-26-39), or low acid levels
('Foster' pink, seedless clone # 1-26-51). Vertical bars denote the SE
of 4 replications and are not pictured where smaller than symbols
used.

Aconitase activity (jumol.g fresh wt . min

97

Fruit age
(month)

3 4 5

6 7

98

Aconitase activity in flavedo and albedo increased dramatically
during the 3rd month of fruit growth (from May to June) and remained
near this level throughout the rest of development (Fig. 6-6). In the
juice tissues, aconitase activity changed in a manner similar to that
of PEPc, MDH, and citrate synthase. Activity was highest in extracts
of the young fruit (May), when acid concentration was greatest.
Aconitase activity from young fruit was less than that of citrate
synthase, and did decrease more rapidly, possibly contributing to acid
accumulation in juice tissues during these periods.

Ratios of citrate synthase to aconitase activity were greatest
from June to July when rates of acid accumulation were most rapid in
both grapefruit clones (Table 6-1).

Discussion

The identification of PEPc activity in grapefruit was not
surprising, due to its presence in a wide variety of tissues, and its
many-faceted function in plants (reviewed by Latzko and Kelly,
1983). Phosphoenolpyruvate carboxylase activity has also been reported
in leaves, roots, and fruit of rough lemon, trifoliate orange, and
'Valencia' orange (Huffaker and Wallace, 1959), and flower buds of
'Valencia' orange (Vu et al^. , 1985).

Of greatest interest, however, is the proposal by Huffaker and
Wallace (1959), that dark CO^ fixation via PEPc may be the source of
organic acids in citrus fruit (Huffaker and Wallace, 1959). Previous
results have indicated that dark CO^ fixation is a major source of
organic acids in citrus fruit (Chapter IV) and that the extent of its
contribution to total fruit acidity becomes more important during

99

Table. 6-1. The rate of organic acid accumulation and ratio of citrate
synthase to aconitase in extracts from ^uice tissues of normal- and
low-acid grapefruit during development.

Grapefruit

clones

Rate of organic acid accumulation
in juice tissues

May

to June June to July

July to Sept

(meq day ^ juice tissue

X)

Normal acidity^

0.25

+ 0.01 0.34 + 0.04

0.20 + 0.06

Low acidity

0.19

+ 0.02 0. 19 + 0.02

0.07 + 0.07

Ratio of

citrate synthase to aconitase activity

May

June July

Sept

(citrate synthase/aconitase

)

Normal acidity

1.41

7.02 3.88

2.51

Low acidity

2.92

4.71 5.29

2.07

z Fruit were harvested in May, June, July, and September after

approximately to 2, 3.5, 5, and 6.5 months' growth, respectively.

^ The normal and low acid grapefruit clones are seedless mutants of
'Foster' pink.

100

development. However, data in the present study show that although
PEPc is quite active in extracts from juice tissues of very young
fruit, activity drops rapidly during development (Fig. 6-3), despite
continued accumulation of organic acids in tissues. Further, PEPc
activities of flavedo and albedo extracts (relatively low acid
tissues) were higher than those from juice tissues (higher acidity) at
most developmental stages (compare Fig. 6-3 and 6-4). The total
activity of PEPc per fruit either in flavedo or in albedo was also
greater than that in juice tissues (data not shown).

Phosphoenolpyruvate carboxylase activity has previously been found not
to coincide with the acid concentration in the tissues of origin
(Clark and Wallace, 1963; Akao and Tsukahara, 1979).
Phosphoenolpyruvate carboxylase may therefore be an important source
of organic acids in citrus fruit (Chapter IV), but is not necessarily
the mechanism controlling accumulation.

A possible function of PEPc in citrus fruit may also be refixation
of respiratory CO^. Recapture of respiratory CC^ by PEPc has been
reported in soybean, pea pod, and tomato (Quebedeaux and Chollet,

1975; Atkins et al. , 1977; Laval-Martin et al. , 1977). Comparison of
CC>2 exchange from intact grapefruit in light and dark indicated that
large amounts of refixation occurred in the light (data not shown).
Although the PEPc activity of flavedo was higher than that of juice
tissues at later stages of development, refixation of CO^ in flavedo
probably occurred via ribulose-biphosphate carboxylase.
Phosphoenolpyruvate carboxylase may predominate for C02 refixation in
the fruit interior. Still, PEPc also acts as a cellular pH-stat and
has maximum activity near pH 8 (Davies, 1973). A CO^ concentration of

101

5% can reportedly lower intracellular pH and affect the enzyme
activity (Bown, 1985). Internal levels of more than 10% were
reported in navel orange fruit 2 days after storage at 20°C (Eaks and
Ludi, 1960). The involvement of PEPc in refixation of respiratory CC^
by juice tissues may therefore be complicated by higher internal CO2
concentrations in citrus fruit.

Formation of fruit acids via PEPc may be associated with the
replenishment of TCA cycle intermediates as suggested by Latzko and
Kelly (1983). This hypothesis also includes a close relationship
between PEPc and MDH activities (Latzko and Jelly, 1983). In vivo
metabolism was not examined in the present study, but assays of
maximal activity for both enzymes showed extensive similarity between
changes in overall capacity of PEPc and MDH (Fig. 6-3 and 6-4). As
noted for PEPc, however, combined activities of PEPc and MDH do not
correspond to acid accumulation within different tissues or to change
in acidity which occur during development.

Citrate synthase has been reported in both mitochondria and

glyoxysomes of maize scutellum (Barbareschi et al. , 1974). In citrus

fruit, however, Vines (1968) found that citrate synthase was active in

mitochondrial fractions, but not detectable in the cytoplasm (cited

from unpublished data in Ting and Attaway, 1971). The existence of the

glyoxylate cycle has been demonstrated in young satsuraa mandarin fruit

14

by feeding sodium glyoxylate-1- C to juice vesicles, but, the rate
was relatively low when compared with that of the TCA cycle (Kobota
and Akao, 1973b). Activities of citrate synthase measured in extracts
from developing citrus fruit (Fig. 6-5) are probably those of the

mitochondrial enzyme.

102

Data on citrate synthase and aconitase in the present paper
provide evidence in support of the following hypothesis. Several
authors have discussed the possibility that combined action of citrate
synthase and the two subsequent enzymes in the TCA cycle, aconitase
and isocitrate dehydrogenase might control the synthesis and
degradation of citrate in citrus fruit (Ramakrishnan and Varma, 1959;
Buslig, 1971; Bruemmer et al. , 1977). Here, results in Fig. 6-5 and
6-6 show, first, that extracts of fruit tissues with high acid levels
had greater activities of citrate synthase relative to aconitase. This
was evident at all stages of development, but was most pronounced
during periods of most extensive acid accumulation (Table 6-1). In
contrast, extracts of tissues which had lower acid levels, flavedo and
albedo, also had less active citrate synthase relative to aconitase at
all but the earliest stage of development examined.

A second line of evidence compared activities of citrate synthase
and aconitase between young fruit of grapefruit clones with normal and
low acid levels. Early in development, low acid fruit was more acid
than that of their otherwise higher acid counterparts. At this time,
citrate synthase activity was significantly higher in extracts of
fruit from the low acid clone, and aconitase activity was lower.
Activities of the two enzymes thus coincided well with initial acid
levels in fruit from both grapefruit clones. Significant differences
in these enzyme activities were not evident between fruit of the two
clones at later stages of growth, but small differences may have
contributed to different long terra acid accumulations.

A relationship between overall efficiency of mitochondrial
metabolism and the acid levels in citrus fruit has been reported in

103

other studies (Bogin and Wallace, 1967; Vines and Metcalf, 1967).

Sweet lemons were found to have more active oxidation and
phosphorylation than sour lemons (Bogin and Wallace, 1967). Citrus
varieties with less fruit acid also showed a higher efficiency in
utilization of respiratory substrates (Buslig, 1971). At less acid
stages, developing citrus fruit more effectively oxidized
intermediates of the TCA cycle (reviewed by Vandercook, 1977). A
partial block in the TCA cycle was presumed to have occurred to a
greater extent in sour than sweet fruit. Studies of satsuma mandarin
juice vesicles indicated that synthesis of citrate and malate was
favored when the TCA cycle was blocked by various inhibitors at
different locations (Kubota and Akao, 1973). Preferential synthesis of
malate or citrate depended on where the TCA cycle was blocked.
Therefore, aconitase may play an important role in sour fruit which
accumulate the higher levels of citrate.

Factors other than the citrate synthase-aconitase enzyme system
cannot be excluded. Enzymes of the TCA cycle are known to be
controlled by many factors such as energy charge, pH, and
compartraentalization of substrates (Wiskich, 1980). The supply of
precursors to TCA intermediates including the amount of carbohydrates
transported from leaves might also be involved as indicated in citrus
by an association between altered glycolysis and shifts in patterns of
acid accumulation (Parekh and Shah, 1971; Parekh et^ al , 1970; Ruffner
and Hawker, 1977).

104

Conclusions

Developmental examination and comparison of enzyme activities in
extracts of flavedo, albedo, and juice vesicles indicated that dark
CO2 fixation via the combined reactions of PEPc and MDH is an
important source of organic acid synthesis, but overall control of
organic acid accumulation apparently exists at steps other than in the
PEPc-MDH pathway.

The greater activity of citrate synthase and lesser activity of
aconitase in tissues with higher acid levels indicated that the
balance between of these two enzymes may influence accumulation by
affecting synthesis and degradation of citrate in citrus fruit.

CHAPTER VII
CONCLUSIONS

14

Distribution of labeled assimilates after light and dark CO^

fixation by leaves and fruit was as follows:

14

1. Export products of both light and dark CO2 fixation in leaves
were deposited primarily in juice sacs of fruit. More of the
assimilates were retained in leaves after dark CO^ fixation.

2. Fruit photosynthesis gave rise to assimilates which remained

primarily in flavedo and albedo 24 h later, thus apparently

contributing little to growth of juice tissues.

14 14

3. C-assimilates from dark CO2 fixation in intact fruit were
localized primarily in juice tissues.

An assessment of the relative C contributed by each assimilate
source to fruit development at various stages of growth indicated that
leaf photosynthesis was most important when fruit were very young. As
fruit expanded, the proportion of C contributed by fruit CC^ fixation
either in light or in the dark increased.

Sources of organic acids were the same as those of total C only in
very young fruit. Leaves were the primary source of assimilates for
organic acid formation early in fruit development. However, dark CC^
fixation by fruit became a major, and perhaps the predominant source
of organic acids later in development, despite its relatively minor
contribution to total C. About 90% of anion fraction of
^C-assirailates recovered from dark fixation in juice tissues of

105

106

5-month-old fruit was citrate and malate. The ratio of labeled citrate
to malate was far less than that of total levels in juice tissues
possibly due to either slow turnover of malate in vacuoles or
continuous refixation in the dark.

Acid concentration decreased and total acidity increased
continously throughout development of fruit from both normal- and
low-acid grapefruit clones, but values were significantly less in
low-acid grapefruit after 5 or more months' growth. In more mature
fruit, the citrate to malate ratio was also significantly less in

fruit from the low- vs. high-acid clone, 9:1 and 14:1, respectively.

14

Significant differences were not evident in amounts of C-assimilates
partitioned into organic acids in fruit of normal- and low-acid
grapefruit clones, but may have resulted from the relatively short
initial exposure time (1 h) compared to the duration of fruit
development.

Developmental examination and comparison of enzyme activities in
extracts of flavedo, albedo, and juice vesicles indicated that dark
CO^ fixation via the combined reactions of PEPc and MDH is an
important source of organic acid synthesis, but overall control of
organic acid accumulation apparently exists at steps other than in the
PEPc-MDH pathway.

The greater activity of citrate synthase and lesser activity of
aconitase in tissues with higher acid levels indicated that the
activities of these two enzymes together may be important to control
of citrate synthesis and degradation of citrate in citrus fruit.

June application of arsenate to calamondin trees or attached,
individual fruit decreased acidity of juice tissues, indicating that

107

arsenical residue in leaves is not necessarily involved in the typical
response to whole-tree sprays. However, effects of arsenate on leaves
and/or fruit peel are likely to be important under field conditions
because arsenical residues in juice tissues do not differ
significantly after arsenate spray (Gilfillan et al. , 1976; Rice et
al. , 1985).

Leaf metabolism appeared to be altered by whole-tree arsenate

treatments in the present study, but not that of fruits. Partitioning

of ^C-assimilates seven days after light and dark ^ CO^ fixation by

leaves and fruits of calamondin trees showed that a greater

percentage of leaf photosynthates was translocated to fruit of

arsenated trees. A larger portion of the total assimilates were thus

found in the organic acid fraction of fruit. Less total photosynthates

were recovered in arsenated trees, however, possibly due to elevated

respiratory rates of arsenate-treated leaves reported by Miller et^ al.

(1933). Percentages of 14C-assirailates partitioned into organic acids

14

did not differ significantly if derived from CO^ fixation by fruit.

The contribution of fruit photosynthesis to growth of calamondin
juice tissues appears to be greater than that of grapefruit (23% vs
less than 4%) and may be related to differences in peel thickness.

LITERATURE CITED

14

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Akao, S. 1979. Translocation to the juice vesicles and changes

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BIOGRAPHICAL SKETCH

Chung-Ruey Yen was born in Taipei, Taiwan, The Republic of China,
on Jan. 5, 1953. He entered the Department of Horticulture, National
Chung-Hsing University in Taichung, Taiwan, in 1971. He received his
Bachelor of Science degree in 1975 and entered the Department of
Horticulture, National Taiwan University, Taipei, for a graduate
program. He studied on postharvest physioloy of 'Bartlett' pear under
the direction of Profs. Ming-nang Chiang and Pin-li Tsai. He received
his Master of Science degree in 1977 and spent another two years as
second lietenant in the army where he was in charge of affairs about
horticulture and administration. After he was discharged from the
army, he got his first horticultural job as a technician and was
responsible for field experiments at the Agricultural Engineering
Research Center in 1980. In order to obtain international experience
in agricultural research and production, he shifted to the Asian
Vegetable Research and Development Center (AVRDC), Tainan, Taiwan, in
1981. He was a research assistant and worked for Dr. Shanmugasundaram
on soybean breeding for 2 years at AVRDC and put much emphasis on
development and breeding of vegetable soybean, a distinctive oriental
protein-rich vegetable. After that, he was an assistant horticulturist
working on tropical and subtropical fruits at Chia— Yi Agricultural
Experiment Station, a branch of Taiwan Agricultural Research
Institute. He was responsible for the breeding of litchi and
passionfruit and management of several tropical fruits such as mango,

119

120

guava, macademia nut, and longan. He entered the Ph.D. program at the
University of Florida in the spring, 1985, under the guidance of Dr.
Karen E. Koch. During this period, he was sponsored a scholarship for
two and half years for Ph.D. study from the Council of Agriculture,
Republic of China, and a graduate assistantship by Fruit Crops
Department. He will receive his degree in December, 1987.

Chung-Ruey Yen was married in 1982 to Li-Lin Chou. Upon completion
of his graduate studies he and his wife will return their country.

Chung-ruey Yen is a life member of Chinese Society for
Horticultural Science. Currently, he is also a member of Intrenational
Society for Horticultural Science, American society for Horticultural
Science, American Society for Plant Physiologist, Florida State
Horticultural Society and California Rare Fruit Growers.

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.

Associate Professor of
Horticultural Science

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.

Frederick S. Davies
Professor of Horticultural
Science

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.

Thomas E. Humphreys
Professor of Horticultural
Science

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.

( i Utot,

C. JackJlearn

Professor of Horticultural

Science

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.

Kenneth J. BootA^/
Professor of Agronomy

This dissertation was submitted to Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of

Philosophy.
December 1987

o

r. js

Dean, CoQ-lege of Agriculture

Dean, Graduate School