METABOLISM OF lODOACETIC ACID
BY ORANGE LEAVES

By

TIMOTHY JOSEPH FACTEAU

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

UNIVERSITY OF FLORIDA
December, 1967

Digitized by tine Internet Arclnive

in 2010 witln funding from

University of Florida, George A. Smathers Libraries with support from Lyrasis and the Sloan Foundation

http://www.archive.org/details/metabolismofiodoOOfact

ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation and gratitude
to Dr. C. H. Hendershott, Division Chairman and Head, Department of
Horticulture, University of Georgia, and chairman of the student's
supervisory committee, for his valuable assistance and guidance of the
research and preparation of this manuscript. He also wishes to express
his gratitude to Dr. R. H. Biggs, Associate Biochemist, Department of
Fruit Crops, and co-chairman of the student's supervisory committee,
for his assistance during the research and preparation of this manuscript.

Appreciation is extended to Dr. A. H. Krezdorn, Chairman, Department
of Fruit Crops; Dr. J. F. Gerber, Associate Professor, Department of
Fruit Crops; and Dr. T. E. Humphreys, Associate Biochemist, Department
of Botany for their constructive criticism and assistance in the
presentation of this manuscript.

The author also wishes to express his deepest gratitude to his
wife, Alice, for her help and thoughtfulness during the course of this
study and the preparation of this manuscript.

11

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF TABLES iv

LIST OF FIGURES vi

INTRODUCTION 1

LITERATURE REVIEW 3

MATERIALS AND METHODS 25

RESULTS 30

DISCUSSION 86

SUMMARY AND CONCLUSIONS 93

LITERATURE CITED 95

111

LIST OF TABLES

1. Effect of the time of exposure to lOAC on the rate of
abscission of 'Pineapple' orange explants 31

2. Effect of various chemicals on the rate of abscission

of 'Pineapple ' orange explants 33

3. Ethanol extractable radioactivity in 'Pineapple' orange
leaf disks treated in 2 ml of 5 x lO'^M IOAC-1-l'^C for

3 hours 38

4. Ethanol extractable radioactivity in extracts of
'Valencia' orange leaf disks treated in 2 ml of

5 X 10-% IOAC-1-l^C for 3 hours 39

5. Radioactivity remaining after ethanol extraction in
'Pineapple' orange leaf disks after treatment in 2 ml

of 5 X lO-'^M IOAC-1-l'^C for 3 hours 40

6. Radioactivity remaining after ethanol extraction in
'Valencia' orange leaf disks after treatment in 2 ml

of 5 X 10-% IOAC-1-l^C for 3 hours 41

7. Distribution of radioactivity in fractions from water
extracts of 'Pineapple' orange leaves treated with
IOAC-1-l'^C 43

8. Distribution of radioactivity in fractions from water
extracts of 'Valencia' orange leaves treated with

IOAC-1-l'^C 44

9. Distribution of radioactivity in fractions of a water
extract of 'Pineapple' orange leaves treated with

IOAC-1-l'^C 45

10. Rf values from paper chromatograms of ^'^C-metabolites
formed by 'Pineapple' and 'Valencia' orange leaves

treated with either IOAC-1-l^C or IOAC-2-l^C 47

11. Distribution of radioactivity in fractions from water
extracts of 'Pineapple' orange leaves treated with

either IOAC-2-l^C or acetate-1-l^C 49

12. Rf values from paper chromatograms of labeled metabolites
formed by 'Pineapple' orange leaves treated with

IOAC-1-l'^C 61

LV

13. Distribution of radioactivity in ethanolic ammonium
and water fractions collected from a Dowex 50-X8

column "^

14. Rf values from paper chromatograms (developed in
butanol solvent) of metabolites formed by 'Pineapple'
and 'Valencia' orange leaves treated with either

IOAC-1-l'^C or IOAC-2-l'^C 70

15. Rf values from paper chromatograms (developed in
methanol solvent) of metabolites formed by
'Pineapple' and 'Valencia' orange leaves treated

with either IOAC-1-l'^C or IOAC-2-l^C 71

16. Rf values from paper chromatograms (developed in
phenol solvent) of metabolites formed by 'Pineapple'
and 'Valencia' orange leaves treated with either

IOAC-1-l'^C or lOAC-Z-l'^C 72

17. Rf values from paper chromatograms (developed in
butanol solvent) of metabolites formed by 'Pineapple'
orange leaves treated with either IOAC-1-l^C or
acetate-1-l'^C 74

18. Rf values from paper chromatograms (developed in
methanol solvent) of metabolites formed by

'Pineapple' orange leaves treated with either
IOAC-1-^'^C or acetate-1-l'^C

75

19. Rf values from paper chromatograms (developed in
phenol solvent) of metabolites formed by 'Pineapple'
orange leaves treated with either IOAC-1-l'^C or
acetate-1-l'^C 76

20. Rf values from paper chromatograms of metabolites
formed by 'Pineapple' orange leaves treated with either
131iOAC or Nal31i 84

21. Distribution of radioactivity in fractions from
'Pineapple' orange leaves treated with sodium

131iodide and eluted from a Dowex 50-X8 column 85

LIST OF FIGURES

1. Effect of the time of exposure to lOAC on the rate of
abscission of 'Pineapple ' orange explants 32

2. Effect of various chemicals on the rate of abscission

of 'Pineapple' orange explants 34

3. ■'•^€02 production from attached 'Pineapple' orange

leaves treated with IOAC-2-14c 35

4. 1^C02 production from detached 'Pineapple' orange

leaves treated with I0AC-l-14c or IOAC-2-14c 36

5. Autoradiogram of thin-layer separated ether-acidic-
partitioned-fraction of 'Pineapple' orange leaves

treated with IOAC-1-l^C 46

6. Autoradiograms of paper chromatographic separation
of I'^C-metabolites from 'Pineapple' and 'Valencia'

orange leaves treated with IOAC-1-l^C 50

7. Autoradiogram of electrophoretic separation of
^^C-metabolites from 'Pineapple' orange leaves

treated with IOAC-1-l^C 51

8. Autoradiogram of ^^C-metabolites from 'Pineapple'

and 'Valencia' orange leaves treated with IOAC-1-^^C 52

9. Electrophoretic separation of I'^C-metabolites
resulting from drop application of IOAC-1-l^C

to 'Pineapple' orange leaves 53

10. Electrophoretic separation of l^C-metabolites
resulting from drop application of IOAC-1-l^C

to 'Valencia' orange leaves 54

11. Electrophoretic separation of 14c-metabolites
resulting from drop application of I0AC-2-14c

to 'Pineapple' orange leaves 55

12. Electrophoretic pattern of 5 x 10"% IOAC-1-l^C 56

13. Electrophoretic separation of l^C-metabolites
resulting from drop application of lOAC-l-^^C

to 'Pineapple' orange leaves 57

VI

14. Autoradiogram of polyamide thin-layer sheet separation
of l^C-metabolites formed by 'Pineapple' orange leaves

treated with IOAC-1-l'^C 60

15, ^^C-metabolites from 'Valencia' orange leaves treated

with IOAC-2-l^C 65

16. l^C-metabolites from 'Pineapple' orange leaves treated

with IOAC-2-l'^C 66

17. l^C-metabolltes from 'Pineapple' orange leaves treated

with either IOAC-1-l^C or acetate-1-l'^C 68

18. I'^C-metabolites from 'Pineapple' orange leaves treated

with IOAC-1-l^C 73

19. l^C-metabolites from 'Pineapple' orange leaves treated

with either IOAC-1-l'^C or acetate-1-l^C 78

20. l^C-metabolites from 'Pineapple' orange leaves treated

with acetate-1-l^C 81

21. l^C-raetabolites of a combined sample of IOAC-1-l'^C and
acetate-1-l'^C each applied by petiole uptake to 'Pineapple'
orange leaves 82

Vll

INTRODUCTION

Increase in citrus production, complicated by a decrease in
available manpower, has resulted in an attempt to develop a mechanical
means of harvesting citrus fruits. The citrus fruit, however, is not
readily adaptable to mechanical harvesting. A major difficulty is the
bonding force between the stem and the fruit which is quite strong,
especially during the early part of the harvesting season. Thus,
investigations into the physiology of abscission were initiated in an
attempt to determine ways to accelerate the abscission processes in
Citrus sinensis cv. Pineapple and Valencia.

It has been shown that field applications of iodoacetic acid
(lOAC) to whole trees resulted in a loosening of the fruit (76, 77).
The lOAC was effective only on early and mid-season varieties ('Hamlin',
'Parson Brown', and 'Pineapple') and not on the late season variety
('Valencia') (76,77). However, both 'Pineapple' and 'Valencia' oranges
could be induced to abscise by lOAC if the compound was absorbed
directly through the stem (168). An investigation of absorption of
lOAC by 'Pineapple' and 'Valencia' orange leaves showed that there was
no difference between these varieties in the amount or rate of uptake
(152). Thus, it would seem that the failure of the 2 varieties to
respond in a similar manner was associated with a difference in
metabolism of the compound. The purpose of the work reported here was
to follow the metabolism of lOAC by 'Pineapple' and 'Valencia' orange
leaves in an effort to determine why the 2 varieties varied in their

2

susceptibilities Co lOAC . It was also hoped that the study would offer
some clue as to how lOAC acts as a promoter of orange abscission.

Another phase of the study involved the use of an explant test to
screen various chemicals for Eheir effects on rates of abaetsslon.
These chemicals were used because they might offer some ideas as to the
mechanism of abscission.

There are 3 possibilities as to how lOAC could act as an abscission
agent. First, lOAC could act as an enzyme inhibitor since it has been
reported as a sulfhydryl enzyme inhibitor in many systems (88). Second,
some metabolite of lOAC , if it were metabolized, could be the active
agent. Finally, the iodine molecule could be the effective part since
it has been shown to be a promotor of abscission (78). This last
possibility would depend on whether or not the I-C bond in lOAC was
broken during metabolism.

LITERATURE REVIEW
Introduction

The process of abscission controls the active shedding of plant
organs. In describing abscission, Esau (53) wrote that the periodic
defoliation of perennial plants is a complex phenomenon which involves
the development of features bringing about the separation. This occurs
without injury to the living tissues and gives protection to the newly
exposed surface from desiccation and invasion by microorganisms.

The morphological and biochemical changes occurring during
abscission are very complex and not completely understood. Also, many
variations in the abscission processes occur among the various plant
species. Some plants form abscission layers, others do not. However,
most plants usually have a distinct zone of specialized cells where
separation occurs (53, 54, 80).

Morphology

The abscission zone is generally located at the base of the subtended
organ such as a fruit, leaf, or flower. The cells within this zone are
usually quite different from the cells in the surrounding areas. They
are usually smaller, denser, and more compact. Intercellular spaces are
absent and there is a conspicuous lack of lignin (3, 53). The cells
usually contain little or no suberin (3) and may or may not be high in
starch (24, 27, 42, 80, 99, 109, 168).

Two layers may be descernible in the abscission zone: a separation
layer, in which structural changes facilitate separation, and a protective
layer, usually believed to protect the plant from desiccation and

3

4
pathogenic invasion (53). Not all plants form a separation layer prior
to abscission (27, 61). An abscission layer was not formed in certain
plants (poinsettia, cotton, pepper) when abscission was accelerated by
ethylene, even though these plants did form a layer prior to abscission
if allowed to develop normally (61). Likewise, in normal abscission of
bean leaves, cell division occurred and an abscission layer was formed;
when abscission was accelerated by ethylene, no cell division took
place and no separation layer was formed (27).

The actual separation process usually requires 2 processes, 1
mechanical and the other biochemical. The actual separation may take
any one of 3 forms: dissolution of the middle lamella, dissolution of
the middle lamella and part of the primary wall, or dissolution of
entire cells (4) .

Environmental Factors

Abscission of plant parts appears to be largely a matter of
biochemical processes. The processes can be modified, and in some
cases initiated, by environmental factors.
Temperature

Abscission processes appear to be temperature dependent since both
high and low temperatures can induce abscission (3). Very high day or
night temperatures have been reported to be detrimental to fruit set of
tomatoes, even with applications of 2-napthoxyacetic acid to inhibit
abscission. It was suggested that the lack of set and subsequent drop
were due to a lack of photosynthates (127). High temperatures also
hastened development of the abscission zones of 'Starking', 'Golden
Delicious', and 'Jonared' apple varieties (146). Low temperatures have
been shown to retard the rates of abscission of bean explants (136).
The response to temperature is thought to be biochemical in nature

5

since it was shown with Che bean explant test that the maximum rate of

abscission occurs at temperatures between 25° and 30° C (174).

Water

Water stress has been shown by many investigators to affect the
abscission processes (44, 81, 113, 146, 165). Early season shedding of
'Washington' navel oranges was reported to be caused by daily water
deficits in young developing fruits (44). However, too much water can
also lead to abscission, since cotton boll shedding was reported
excessive if the root zone became flooded (51).
Light

Light intensity, duration, and quality have been shown to effect
abscission and some investigators (71) are of the opinion that abscission
is not entirely an auxin-mediated response. With light-grown seedlings,
chemical treatments had a more pronounced effect upon abscission than
did dark-grown seedlings. Light quality was shown to have just as
significant an affect upon abscission as it did with dark-grown
seedlings. It was suggested that high light intensity reduced abscission
probably because of rapid dehydration and enzyme inactivation (71).
Other workers (15) showed that light had an inhibitory effect on the
rate of abscission of young bean explants. However, the effect diminished
as the plants aged.

Internal Factors
Effects >jI Auxins on Abscission

The role of auxin in abscission of leaves had been recognized ever
since Laibach (94) found that auxin-rich orchid pollinia would both
accelerate and retard the abscission of debladed petioles. LaRue (97)
and Portheim (130) were also instrumental in establishing that auxins
applied to leaf petioles delayed abscission. Since then, there have

6

been raany reports on Che action of auxin in relation Co the abscission
proce-j (1, 3, 4, 13, 15, 16, 102, 136, 155).

One of Che early cheories on Che action of auxin was Che "auxin-
gradient" Cheory (144). Work with beans established the facts tlmt
levels of leaf auxin were higher than levels of sCalk auxin and this
"gradient" decreased with age. From these facts, the idea arose that
an auxin gradient controlled abscission. The theory received criticism
from various workers (16, 60, 119, 150, 153) who found that auxin
applications either distal or proximal to Che abscission zone were
effective in delaying abscission.

The concentration applied to plants has been shown Co influence
abscission. High concentrations of auxin applied to coleus and bean
explants have been shown to inhibit and low concentrations Co
accelerate abscission. WheCher the applications were proximal or
disCal to the abscission zone made no difference . (60) . These results
were confirmed (13, 16) and the two-phase theory of Che acCion of auxin
on abscission was proposed.

Furcher work revealed the existence of a time factor (41, 102, 136,
138, 141). Auxin applied, in any concentration, within 6 hours after
deblading delayed abscission. After this, all concentrations of auxin
accelerated abscission proportionally to the concentration applied. The
initial period (delayed by auxin) was called Stage I, and Che second
(acceleraced by auxin) was called SCage II.
Effects of Auxins on Pectin Substances

Since abscission involves dissolution of pectin compounds and/or
cell walls, investigations have been made of the effects of auxins on
these materials. The pectic substances are primarily polymers of
galacturonic acid and act as cellular cementing agents. They are Che

7

basic components of the middle lamella (53). The carboxyl groups
present are bonded through calcium and magnesium ions to other chains,
thus binding one cell to another. Methylation of the carboxyl groups
probably reduces the bonding strength of the pectic substances (23, 56,
122, 154).

The literature regarding pectin enzymes is confusing. There are a
number of enzymes involved with pectic compounds; i.e., pectinase which
hydrolyzes pectic acid, but not methylated pectic acid (175); poly-
galacturonidase which hydrolyzes pectin chains (175); and pectin methyl-
esterase (PME) that de-es terif ies carboxyl groups (102).

It has been noted that soluble pectins increased as apple fruits
ripened and this was attributed to the action of PME and/or polygalac-
turonidase (102). Both enzymes have been found in the abscission zones
of debladed bean petioles and it was suggested that PME was necessary
for free carboxyl groups so that polygalacturonidase could split the
long pectin chains (133). High PME activity has been reported (171-
173) in the abscission regions of tobacco pedicles. From tests with
indoleacetic acid (lAA) and methionine (methyl donor), it was concluded
that abscission was prevented by high PME activity and increased by low
PME activity. In agreement with this, highest PME activities have been
found to occur in the abscission zones of non-abscissing leaves (95).
Thus, PME activity may be associated with leaf age since high PME
activity has been reported to occur in young bean abscission zones and
to decrease with age. When abscission was stimulated by ethylene, a
decrease in PME activity occurred. Moreover, treatment with 2 ,4-dichloro-
phenoxyacetic acid (2,4-D) inhibited abscission and the PME activity
remained high. It was suggested that the de-esterif ication of methyl
groups caused by the high PME activity in the presence of 2,4-D would

8

serve to make sites available for calcium binding, thereby strengthening
the cell walls and inhibiting abscission (126).

The addition of lAA has resulted in an accelerated rate of methyl
es terif ication of pectic substances in cell walls of Avena coleopciles,
but has not resulted in a net change in the final degree of pectic
esterif ication (89). Also, lAA increased PME activity in tobacco pith
cells and these results were used to explain the increase in growth.
The suggestion was that removal of methyl groups by PME allowed a
polygalacturonase to further break down pectin, thus producing
elasticity and, thereby, an increase in cell enlargement (29). Further-
more, lAA may promote the bonding of PME to the cell wall, thus tending
to immobilize the enzyme and favor the methylation of pectates (or
prevent de-esterif ication) , which would decrease the amount of calcium
bridging, thereby, causing softening of cell walls (65). Auxin applications
have been reported (124) to increase the incorporation of the methyl
group from methionine into pectins of Avena coleoptile cell walls, thus
softening them. Moreover, anti-auxins have been found to inhibit the
lAA effect of loosening cell walls (43).
Effects of Ethylene on Abscission

The ability of ethylene to accelerate abscission has long been
recognized (1, 3, 13, 27, 32, 61, 67, 68, 72, 108, 116, 137, 139, 147).
However, other unsaturated hydrocarbons can produce the same effects as
ethylene, but are generally required in higher concentrations (3).
Whether ethylene, per se , is the cause of naturally occurring abscission
processes is not known. It may be a by-product of catabolism and does
not initiate the abscission process, but may simply speed it. If the
biosynthesis of ethylene were known, the problem would be simpler. For
instance, if pectin substances are sources of precursors to ethylene

9

produ^ cion as suggested (67), then ethylene would probably be a by-
product of pectin breakdown.
Effect of Chernical Treatments on Ethylene Production

The discovery that leaves produced ethylene, that this production
increased as abscission advanced, and that exogenous lAA could inhibit
this increase, led to the auxin-ethylene balance hypothesis of foliar
abscission as proposed by Hall (67, 68). It was shown that arabinose,
ethanol, pectin, pectic acid, pyruvic acid, fructose, and galactose
yielded ethylene (67).

However, 2,4-D and lAA have also been reported to stimulate the
release of ethylene by cotton plants (117, 118) and from bean explants
(1, 137). Yet these compounds, under certain circumstances, retard
abscission. Addicott (3) also concluded that ethylene probably functions
in abscission through its effects on auxin.

Many compounds are known to influence abscission, auxin being an
endogenous regulator. Besides auxin, treatment of bean explants with
endothol, potassium iodide, and some amino acids result in increased
rates of abscission plus an increase in ethylene production (1, 137).
In fact, all chemical agents that stimulated abscission only did so if
applied during Stage II. Ethylene, the most potent chemical, had no
effect except during Stage II (137). Conditions in which ethylene would
not build up were used and decreased rates of abscission were found.
These facts led to the conclusion that ethylene was involved in the
abscission process (137).
Possible Mechanisms of Ethylene Biosynthesis

One of the more basic problems involved with ethylene is the mechanism
of its synthesis in living plants. Ethylene can be found in most plant
parts, especially ripening fruits (31, 33, 34, 104, 105, 110, 114). It

10

has been shown to be increased by additions of auxin (1, 3, 67, 70),
abscission agents (1, 137) and to be formed from many substrates
present in plants, including pectin compounds (67, 70).

The search for the pathway of ethylene production has led to the
separation of various sub-cellular systems. This search was instigated
because it was observed that intact tissues respond to treatment with
solutions of varying tonicity as though the ethylene-producing system
was located in a particle having a semi-permeable membrane. Cytoplasmic
particles that would evolve ethylene in the presence of thiomalic and
thioglycolic acids have been isolated. The system had many character-
istics of an enzyme system in that ethylene production was proportional
to the concentration of particles, the reaction was stopped by heat,
and increased by phosphorous. Ethylenediaminetetra acetic acid (EDTA)
inhibited the reaction and this inhibition was partly reversed by
adding copper (104).

Attempts to repeat this work have led to the conclusion that the
substances emanating from the cytoplasmic particles was not ethylene
and, hence, ethylene production by a sub-cellular system had not yet
been found. The gas that was found reacted similarly to that previously
isolated, but it did not co-chromatograph with ethylene. Neither bromine
nor mercuric perchlorate solutions removed the substance from air,
whereas, these reagents were found consistently to eliminate comparable
quantities of ethylene from synthetic air-ethylene mixtures. The gas
chromatographed between ethane and ethylene and might have been a 2-
carbon compound (31).

Still other workers concluded that the gas in question was ethane
(105). This ethane-producing particulate system required the presence
of an unsaturated fatty acid. Saturated fatty acids gave little to no

11

production of ethylene, while linoleate and linolenate resulted in a
marked production of ethylene. It was not clear whether these acids
were acting as co-substrates or co-factors. Under normal conditions
apples usually produce more ethylene than ethane, and if apple tissue
was homogenized and incubated in buffer, the 2 were produced in equal
amounts. Under these same conditions, ethane was produced from thiomalic
acid. From these relations it was suggested that a possible relationship
existed between ethylene and ethane biosynthesis and that present
information suggests either one may be a precursor of the other, or
they are derived from a common source (105).

Mitochondria may be involved in the synthesis of ethylene since
2,4-D treated cotton plants responded by an increase in both CO2 and
ethylene production, all of which occurred in the mitochondria (117).

Buhler e_t al. (30) subjected various fruits to ethylene-l^C and
found that avocados and pears incorporated l^C from ethylene, but oranges
did not. The amount of l^C incorporated, in any case, was very small.
The majority of the radioactivity was in the organic acid fraction,
suggesting that ethylene was metabolized through the organic acid cycle.

However, mitochondria preparations from both tomato and apple fruits
did not produce ethylene, indicating that ethylene production and the
Krebs cycle were not connected (114). Other workers (104) also concluded
that ethylene synthesis was not involved with the Krebs cycle since
preparations which evolved ethylene were not mitochondria fractions
and would not oxidize Krebs cycle substrates.

Apple slices have been shown to produce labeled ethylene when
treated with tritium labeled water. The optimum temperature for the
process was 32° c and above this temperature ethylene production decreased
rapidly. The inactivation caused by heat slowly disappeared when the

12

tissues were exposed to lower temperatures. Also, ethylene synthesis
ceased almost immediately under anaerobic conditions, but a precursor
accumulated that could be rapidly oxidized in air to yield ethylene (33).
Effects of Gibberellins on Abscission

Gibberellic acid (GA) has been noted to affect abscission. Increased
abscission rates after application of GA have been obtained by numerous
investigators (1, 2, 13, 24, 39, 40, 79, 93, 119) and some (38)
hypothesized that 3 hormones, auxin, GA, and an abscission-accelerating
hormone, interact to control the process. The mechanism of a GA-auxin
interaction, if present, is unknown. Treatment with GA has been shown
to increase levels of endogenous auxin in plants, possibly by influencing
the lAA degradation enzymes, peroxidase and lAA oxidase, either directly
or through the action of an inhibitor (66). That GA directly or
indirectly controls the endogenous level of auxin has also been
concluded by others (128). However, based on the facts that GA
promotes growth under optimal concentrations of auxin and that GA and
auxin have opposite effects on cell walls, Leopold (102) suggested that
GA and auxin act through distinct and separate systems.

GA has been shown to stimulate abscission in Stages I and II of
bean explants (40), but it was most active in Stage I. However, GA also
exerts the same two-phase concentration action as does auxin as it
prevents abscission at high concentrations, but stimulates abscission
at low concentrations (38). GA also has been shown to stimulate ethylene
production (1).
Effects of Kinins on Abscission

The opinion exists (162) that kinins are the predominant regulators
in the early part of fruit development following fruit set. It has been
suggested (101, 102) that kinins influence cell division and synthesis
of protein, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA),

13

Furthermore, kinins can influence the abscission process (40, 128).
Some investigators (40) found that kinetins exerted the same two-phase
action as did auxin and delayed abscission in Stage I. However, after
Stage II was reached, it too stimulated abscission. Others (128)
have shown that applications of kinetin to the abscission zone inhibit
abscission, but either proximal or distal applications accelerate
abscission. Applications of kinetin to the abscission zone caused
cellular activity and movement of metabolites into that region, thus
preventing senescence and abscission. Other investigators (143) have
confirmed that mobilization of metabolites into the abscission zone
could defer senescence. Increases in dry weight, chlorophyll content,
protein, DNA, and free and total phosphates were found proximal to the
abscission zone in bean explants and whole leaves when kinetin was
applied proximal to the abscission zone (143).
Effect of Endogenous Abscission Regulators on Abscission

It has been noted (125) that as bean leaves became senescent and
approached abscission, the diffusate into agar blocks from these leaves
was progressively more effective in accelerating abscission. Subsequent
work by various investigators (13, 14, 69, 87, 108, 163) revealed the
presence of other abscission regulators in other plants. In 1961, Liu
and Carns (107) crystallized an abscission accelerating material from the
cotton burr which they named "abscisin". This regulator was readily
translocated and inhibited the retardation of abscission induced by
lAA. Others (5) have extracted 2 different regulators from cotton; 1
from young bolls (abscisin II) and 1 from older bolls (abscisin I).
Further work with abscisin II revealed that this regulator accelerated
petiole abscission of beans, citrus, and coleus , as well as cotton (6).
It also accelerated senescence (yellowing) in detached radish leaves.

14

Furthermore, the regulator also counteracted the effects of lAA in the
Avena curvature and straight-growth test, and GA in the dwarf maize,
dwarf pea, and barley endosperm bioassays.
Effects of Carbohydrates and the C-N Balance in Relation to Abscission

It has long been thought that a low content of carbohydrates leads
to leaf, flower bud, and fruit abscission (4). Various investigators
have reported that the addition of sucrose (13, 15, 27, 96, 108) or
high tissue levels of carbohydrates resulted in delays in abscission
(41, 49, 74, 108).

In contrast to these findings, Eaton and Ergle (52) concluded that
the nutritional theory of boll shedding of cotton was not valid with
regards to carbohydrate and nitrogen relations. Within varieties and
environments, the number of bolls/100 g fresh steins and leaves remained
constant even though nutritional factors caused marked differences in
plant growth.
Effects of Amino Acids on Abscission

Methionine and certain other methyl group donors have been shown to
be effective in accelerating abscission of tobacco flowers and petioles
of cotton and coleus (112, 161, 172, 173). The use of l^C labeled
methionine and phenylalanine indicated (161) that methionine and
phenylalanine might promote abscission by serving as sources of methyl
or other groups which could be incorporated into the cell wall and
middle lamella in the separation zone. The D forms of alanine, aspartic
acid, glutamic acid, and serine were found to be effective as promotors
of abscission (161). Also, both the D and L forms of leucine, methioniiie
and phenylalanine had some activity (161). However, other work has
shown that the most efficient methyl donating compounds were, in general,
far less effective in accelerating abscission than were some other
compounds (140).

15

Alanine has been reported to increase abscission (41, 136, 137, 140)
and alanine and some other amino acids can result in increased ethylene
production in the bean explant test (137). It has also been suggested
that as a leaf ages, the decline in auxin along with a concomitant rise
in amino acid concentrations could promote abscission (41). Since
kinetin increases the synthesis of DNA, RNA, and protein, and since it
will inhibit abscission when placed on the abscission zone of bean
explants (128), it would seem that amino acids could conceivably play a
part in the abscission process. Amino acid extracts from various aged
leaves do suggest a relationship with the abscission process as extracts
iirom older leaves accelerated the rate of abscission (140).

Biochemistry of Abscission

Deficiencies of oxygen (39, 108), carbohydrates (13, 15), water
(69), and growth regulators (3, 139) can promote the initiation of the
processes leading to separation. The exact nature of the functioning
of any one of the factors in unknown. Furthermore, the processes leading
to senescence may in some way be connected to the processes of abscission.
Generally, in the literature, the 2 terms are difficult to separate.
Abscission of plant parts can occur from relatively young plants. How-
ever, this does not mean that the part abscissed was, in turn, physiolog-
ically young. The results of experiments utilizing kinins , suggest that
senescence and abscission are interrelated. Kinetin causes a mobilization
of organic compounds and an increase in protein, DNA, and RNA synthesis.
Abscission is generally associated with a loss of carbohydrates and a
mobilization away from the abscission region. Also, as leaves approach
senescence, the inhibitory effects of auxin are lost.

The physical changes occurring in senescent (or abscissing) leaves
are very evident, i.e., chlorophyll and water loss, and anthocyanin

16

appearance. The chemical changes include exit of nitrogen, potassium,
phosphorous, iron, and magnesium, changes in form or disappearance of
carbohydrates, and a decrease in the auxin level (3). Just exactly
why tiiese changes occur is unknown, but many theories have been advanced,
among which are the accumulation of some inhibitor or deleterious
substance, the accumulation and deposition of calcium, and permeability
changes in membranes (164).
Changes in Glucose Metabolism as Tissues Age

There is evidence which indicates that as plants grow older, a
shift in their various metabolic pathways may occur. Differences in
sensitivities to metabolic inhibitors have been found between young
and old plants, which would imply at least a difference in basic
metabolism (111). Also, in young tissues and in undifferentiated
tissues, the glycolytic pathway was of major importance. However, as
the tissues aged the pentose shunt was favored (12). Changes of
sufficient magnitudes in certain enzymes have been reported to provide
a convincing explanation for the change in pathways as tissues age (64).
Moreover, the presence of the pentose shunt in fruits of peppers (50),
tomatoes, cucumbers, limes, and oranges has been demonstrated, but the
authors did not study changes with age (7).

Thus, workers have provided evidence that a shift in the method
of glucose degradation occurs as plants age. In contrast, there are
indications that shift from the pentose shunt to the glycolytic pathway
occurs in ripening banana fruits (151).
Possible Relationships of the Pentose Shunt and the Abscission Processes

Glucose catabolism appears to shift from the glycolytic pathway to
the pentose shunt as tissues age (12, 111). Since leaf senescence and
abscission are closely related, with abscission possibly being the

17

terminal process, the pentose shunt may be associated with these
processes. Auxins (82, 83, 141), as well as lOAC (8-10, 55, 90),
influence the pathway of glucose catabolism; moreover, both are
involved with abscission (3, 4, 37, 73, 76, 77, 98, 102, 136, 167-169).

It has been suggested that lOAC-induced abscission of orange
fruits might involve a shift to the pentose shunt (168). lOAC blocks
glycolysis at the triosphosphate dehydrogenase (TPD) step (11, 23, 35,
55, 63, 90) and it would seem likely that an inhibition at this point
could cause increased activity of the pentose shunt. This has been
shown to be the case in chlorella (90) and in apple slices (55), but
not the case for strawberry leaves (8-10).

Auxins, especially 2,4-D have been found to influence the pathway
of glucose catabolism. An increase in the pentose shunt with 2,4-D
treatment has been reported', but lAA had no effect (82, 83). Others
(142) found that lAA or kinetin reduced the activity of gluconate-6-
phosphate dehydrogenase and transketolase to only 1/3 to 1/4 that of
the controls. The activities of enolase, malate dehydrogenase, and
isocitrate dehydrogenase were not affected by the presence of lAA or
kinetin. So, with increasing growth rate (tumor tissue), there was a
decrease in the activities of the pentose shunt. However, it has also
been reported that additions of auxin resulted in an increase in glucose-
6-phosphate dehydrogenase activity (91).
Effects of lOAC on Abscission

lodoacetic acid has been shown to accelerate abscission in oranges
(76, 77, 168, 169), olives (73), bean plants (167), and cotton explants
(37). Weintraub e_t a_l. (167) surveyed over 500 compounds on bean plants
to see if they would accelerate abscission. Triiodobenzoic acid was
used as a standard. The activity of related compounds was influenced

18

by Che halogen, iVBr^Cl (in order of the most to the least effective
inducing abscission) and by specific position occupied (3^2 or 5).
lodoacetic acid was also found to cause abscission but to a lesser
extent than triiodobenzoic acid.
Effects of lOAC on Biochemical Systems

lodoacetic acid is known as an inhibitor of certain enzymes,
particularly sulfhydral (SH) enzymes (23, 88, 100, 103, 115, 156, 157).
In some cases, the primary enzyme affected was TPD (88), but others (35)
found that lOAC would inhibit CO2 fixation but did not inhibit TPD. In
contrast, it was reported that lOAC inhibited both CO2 fixation and TPD
activity in chlorella (90). Under conditions of darkness, increased
levels of fructose 1 ,6-diphosphate (FDP) , dihydroacetone phosphate (DAP),
and glyceraldehyde phosphate (GAP) resulted. However, when the chlorella
were treated with lOAC in the light, the effects of lOAC disappeared.
It was suggested that in darkness, lOAC inhibited TPD while FDP, DAP,
and GAP accumulated. In the light, FDP was converted to ribulose 1,5-
diphosphate (RUDP) with the help of cyclic phosphorylation and by
carboxylation to the B keto acid in the pentose shunt. lOAC , therefore,
induced a new pathway of hexose degradation via RUDP (90). lOAC also has
been shown to promote an increase in anthocyanin in 'Mcintosh' apples.
It was postulated that lOAC decreased glycolysis and increased glucose
metabolism via the pentose shunt, thereby increasing the shikimic acid
concentration and leading to the production of more anthocyanins .

However, it was shown that lOAC stimulates glycolysis to a much
greater extent than it stimulates the pentose shunt. Strawberry leaves,
when treated with lOAC , responded by large increases in CO2 production
which was not completely accounted for by losses of sugars and starches.
This increase in CO2 production, however, was associated with a rise in
the concentrations of pyruvate and oxaloacetate (9, 10).

19

Further studies of the C5/C1 ratio in strawberry leaves treated
with lOAC indicated that increased glycolysis accounted for the major
part of the stimulation of CO2 output. Glucose-6-phosphate (G6P),
fruc tose-6-phosphate (F6P), and FDP increased greatly. The increases
in G6P and F6P, caused by iodoacetate, were attributed to increased cell
wall permeability such chat there was an increase tn the accessibility
of enzymes to substrates. It was suggested (10) that some of the
increased C02 production was partly caused by uncoupling of oxidative
phosphorylation since Contreiras (47) postulated that lOAC acted as an
uncoupler of high-energy phosphate bonds.

lodoacetamide , 2 ,4-dinitrophenol (DNP) , fluoride, arsenite, sodium
bisulfite, and f luoroacetate all were shown to inhibit ethylene production
and respiration in apple tissues (34). The inhibitions caused by DNP
were j .irtially reversed by adenosine triphosphate (ATP). The evidence
suggested that at least 1 step in the synthesis of ethylene required
energy which was supplied by respiration. Another step might involve
a sulfhydryl enzyme since high-energy compounds failed to reverse the
inhibitory effects of lodoacetamide.

Whether lOAC , as do other abscissing agents including potassium
iodide (137), causes an increase in ethylene production which initiates
abscission; or whether lOAC inhibits ethylene production and, therefore,
abscission accelerated by ethylene, remains to be investigated. Also,
lOAC might act as a SH inhibitor. Michaeles and Schubert (115)
postulated that the reaction R-SH-l-ICH2C00H-> R S-CH2COOH :-HI was the
mechanism by which lOAC affects SH enzymes. However, Wilson (168) tried
unsuccessfully to histologically determine the effects of lOAC on SH
enzymes in the orange fruit abscission zone.

20

Sweet orange leaves are able to decarboxylate IOAC-l--'-'^C . It was
not ascertained: a) whether the lOAC acted before the carboxyl group
was split off; b) whether the iodine, per se, induced abscission; c)
or whether the methyl carbon alone as the ICH3 moiety was the effective
part (152).
Effects of Potassium Iodide on Abscission

Potassium iodide has been shown to be capable of accelerating
abscission of bean leaves. Moreover, applications of lAA inhibited
the effects of the iodide ion. It was also shown that iodine also
would accelerate bean leaf abscission. Defoliation of immature cotton
required 1000 times as much iodide ion as did beans, indicating that the
concentration required to accelerate abscission varies with species (78).
The effectiveness of other halogen ions with respect to accelerating
abscission has been tested. It was shown that iodine was more effective
in accelerating abscission of bean leaves (167) and orange fruit (76,
168) than was bromine, chlorine, or fluorine. Potassium iodide also
is active on deciduous plants (98).

Whether the iodine in potassium iodide functions, in abscission in
a similar manner as does the iodine of lOAC is not known. Rubinstein
and Abeles (137) showed that potassium iodide accelerated the abscission
of bean explants, but an increase in ethylene production occurred before
tissue separation. This led to the conclusion that potassium iodide and
other abscission promoters acted through their effects on ethylene
production. The effects of lOAC on ethylene production are not known,
but iodoacetamide has been shown to inhibit ethylene production in apple
slices (34) .
Effects of Oxidation-Reduction Agents in Relation to Abscission

Growth promotion of stem tissues of cucumbers, induced by auxin.

21

has been associated with an increase in ascorbic acid and a more reduced
state; whereas growth inhibition of leaf tissue has been associated with
a decrease in ascorbic acid and a more oxidized system (91). The auxin
treatments resulted in an increase in glucose-6-phosphate dehydrogenase
activity and also an increase in nicotinamide adenine dinucleotide
phosphate (NADP) production . It was suggested that the NADPH2 produced
could lead to a more reduced state of the glutathione and ascorbic acid
systems. Several workers (112, 145, 159) believe that these 2 systems
are important factors in growth.

In contrast, however, no significant connection was found (106)
between the effects of auxin and the ascorbic acid-dehydroascorbic acid.
These same investigators found no increase or decrease in ascorbic acid
or dehydroascorbic acid with additions of auxin. Therefore, the dehydro-
ascorbic acid was not the factor that resulted in the reduced growth.

Ascorbic acid has been reported to induce abscission of oranges
when applied in high (2-57o) concentrations to the leaves as a dip (48).
The ascorbic acid only promoted abscission when applied to the leaf
tissue, not when applied to the fruit itself, suggesting that some
metabolite or change in the ascorbic acid was responsible for the action.
A difference in uptake might also have been involved since leaf tissue
would probably absorb more ascorbic acid than would the orange fruit.

Reducing agents, such as bisulfite, cupric and ferric ions, have
been shown to promote orange abscission. It was thought that possibly
the process of abscission was involved with keeping tissues in a reduced
state (168, 169). However, it has been proposed (145) that the onset
of senescence is controlled by an unfavorable pile-up of oxidants
(electron acceptors) upsetting an endogenous antioxidant-oxidant
balance (acceptor/donor ratio). This suggests that abscission, a

22

manifestation of leaf senescence, might be retarded by conditions that
tend to favor the preservation of a more juvenile and less oxidized
(more reduced) state. This idea is contrary to the previous one
concerning abscission (163, 169). As evidence, it was reported that
lAA and other antioxidants (electron donors) inhibit lignin synthesis
which is usually a process carried out in older tissues (145).

Auxins are known to be effective in either inhibition or accelerating
abscission of many plants. Whether or not auxin is effective in enhancing
orange fruit abscission is uncertain, but 2,4-D definitely acts as an
inhibitor (168). Whether the action of auxin on abscission is through
the ox i-dation-reduc tion state of the tissues is not known.

Acetate Metabolism
Aromatic Synthesis

Acetate has been shown to be involved in the biosynthesis of many
aromatic compounds (120, 121, 135, 158, 160). There appear to be 3
general modes of incorporation: a) from acetic acid by head-to-tail
placement in a straight chain with additions of Cl units from the C^
pool, b) from head-to-tail placement via condensation, and c) from the
isoprene route similar to b) , but with an intermediate similar to or
being mevalonic acid (18). However, the shikimic acid pathway also is
a major pathway of aromatic biosynthesis (120). Protocatechur ic , gallic,
cinnamic acid derivatives, coumarins , and others have been shown to be
formed from a shikimic acid pathway (120JI Also, there are other aromatics
that are formed by a combination of acetate and shikimate pathways, among
which are flavonoids, isoflavones, and isocoumarins (12, 121).

Birch e_t_ al. (17) were the first to show that fungal cultures could
form an aromatic compound from acetate-l-^^C according to the head-to-
tail condensation theory proposed by Birch and Donovan (18). Since then,

23

others (19-21, 57-59, 120, 134) have demonstrated that many aromatic
compounds are derived from a head-to-tail condensation. The mechanism
in all these cases seems to be related to fatty acid synthesis (120).
However, it was reported (20) that methyl groups in 7-hydroxy-4, 6-
dimethylphchalide were derived both from methionine and from the methyl
carbon of acetate. Formic acid also contributes C]^ units to some
phenolic compounds (20, 21).

Another pathway by which acetate is incorporated into aromatic
rings is via mevalonic acid (120, 121). Some benzene rings are formed
thusly, but assimulated acetate also goes into side chains. Heinstein
et al ■ (75) studied incorporation of labeled acetate into gossypol in
excised cotton roots and concluded that the mevalonate pathway was
probably the pathway of biosynthesis.

It has been suggested (120) that shikimic acid could serve as a
precursor to many types of aromatic compounds. Evidence to support this
comes from many sources (120, 121). The synthesis of the B ring of
quercetin, synthesized in buckwheat (Fagopyrum tataricum), was found to
be derived from shikimic acid (160) while the A ring and carbons 2, 3,
and 4 of C]^5 flavonoid compounds were derived from acetate (62, 166).
Caffeic acid in buckwheat and tobacco was derived solely from phenylalanine
(62). Likewise, phloridzin biosynthesis in Malus (84) and hydrangenol
in Hydrangea (85, 86) are derived by similar pathways. However, there
are groups of aromatics that are derived only from shikimate, since
coumarins were reportedly formed by the shikimic acid pathway and acetate
was poorly utilized (28). Also cinnamic acid derivatives have been
noted to arise mainly from shikimate (120).

24
Fatty Acid Synthesis

Acetate is metabolized to fatty acid compounds via malonyl-COA
(92, 120, 149). The reactions are now fairly well established and
have been demonstrated in cell-free systems (25).
Isoprenoid Synthesis

Isoprenoid structures are alr.o derived from acetate via a head-
to-tail condensation (129, 170). The pathway has been elucidated,
mainly in mammalian tissues, because cholesterol and many animal
hormones are formed via this pathway. However, phenolics formed by
this pathway have been found in plants (75).
Glyoxylic Cycle

The glyoxylate cycle is another means by which acetate can be
metabolized. Acetate feeding experiments, mainly with fatty materials
(i.e., castor beans) have shown that acetate is incorporated into citric
and malic acids in the Krebs cycle (36). The 2 key enzymes, raalate
synthetase and isocitritase , occur in many micro-organisms and plants,
particularly in high oil seeds. Seeds depending on starches for energy
rather than fats do not possess the glyoxylate cycle, so it may not be
universally present (46).

MATERIALS AND METHODS
Plant: Material

All experiments were conducted with either leaves or fruits from
'Pineapple' and 'Valencia' sweet oranges. Two-year-old trees started
from cuttings and grown in containers were used for tests in which the
radioactive materials were applied in localized zones. Orchard trees
were used in experiments in which a petiole absorption technique was
employed .

The orange explant test was used to screen chemicals for their
abscission accelerating abilities as described by Wilson (168, 169).
Briefly, the explant consisted of an orange fruit with a 3-4 inch stem.
The stem was inserted into the test solution for the duration of the
test. To determine if abscission was in the final stage, a force was
applied to the abscission zone by applying a slight pressure to the
side of the stem.

Determination of C02 Production
Continuous Flow System

The production of ^^002 from intact 'Pineapple' orange leaves
treated with I0AC-2-l4c was determined by trapping 1^002 in Hydroxide
of Hyamine 10-X p- (diisobutyl-cresoxyethoxyethyl)-dimethylbenzyl-
ammonium hydroxide (132). The leaf, still attached to the plant, was
sealed in a small plexiglass leaf chamber.- The method of collecting
■'■^002 evolved by treated leaves was via a scrubbing train. Air was
forced through a train of a scrubbing tower of 10% sodium hydroxide, a
distilled water washing tower, a plexiglass leaf chamber, and, finally,

25

26
through the trappiog solution.
Closed System

After treatment with either IOAC-1-l'^C or IOAC-2-l^C, 2 'Pineapple'
orange leaves, still attached to approximately 3 inches of twig, were
placed so that the proximal inch of the twig was in water. The container
plus water and explants were suspended in a 250 ml Erlenmyer flask in
which 20 ml of Hydroxide of Hyaraine was added to absorb C02- One ml of
the trapping solution was removed (and 1 ml replaced) with a syringe
and hypodermic needle through a vaccine cap attached to a teflon tube
leading below the level of the trapping solution.

Radioactive Materials and Methods of Determining Radioactivity

All radioactive materials were obtained from the Nuclear Equipment
Company at the following specific activities:

IOAC-1-l^C, sodium salt, 1.4 mc/m mole
I0AC-2-14c, sodium salt, 6.5 mc/m mole
Acetate-1-l^C , sodium salt, 15 mc/m mole
Nal31l, sodium salt, 555.5 mc/m mole
Water was added to make concentrations of O.IM lOAC and 3 x 10"% acetic
acid .

^^^IQAC was prepared essentially as described by Conant and Kirner
(45). Nal31x was reacted with 100 mg of monochloroacetic acid in 10 ml
of refluxed, redistilled acetone. The reaction took place at 40° C in
a sealed bottle for 20 hours. Acetone was removed under a stream of
nitrogen and the residue was washed with 15 ml of carbon disulfide.
The remaining residue was dissolved in 0.5 ml of deionized water.

Both liquid scintillation and Geiger-Mueller methods of determining
radioactivity were used. A Packard-Tri-Carb liquid scintillation counter
series 314 Ex at -5° C was employed with 2 counting solutions: a) a

27

solution for counting the Hydroxide of Hyamine; 2,4-diphenyloxazole (PPO)
3 g; 2,2' -paraphenylene bis 5-phenyloxazole (POPOP) 100 mg; toluene
1000 ml (132); and b) a solution for counting high percent water samples
as described by Bray (26), namely, PPO (4 g) POPOP (0.2 g) naphthalene
(60 g) methanol (absolute, 100 ml) ethylene glycol (20 ml) and p-dioxane
(to make 1 liter). All planchet counting was conducted with a Nuclear
Chicago GM planchet counting system. The instrument used was a model
183-B Count-0-Matic scaler equipped with a model C-lll-B time-interval
printer, a model C-llO-B automatic sample changer and, a model D-47 gas
flow detector.

Paper chromatograms and paper electrophoretograms were scanned
using an Analytical counter ratemeter model 1620-B, equipped with a
Nuclear Chicago model C-IOO-A Actinograph II and a D-47 window gas
flow detector. Recordings of the scan were made with a 1-MA Esterline-
Angus chart recorder. Settings used were: slit width, 1/4 inch, time
constant 10 seconds, full scale deflection 150 or 300 cpm, and chromato-
gram scan speed of 3/4 inch/min.

Radioactive samples eluted from polystyrene column were monitored
by passing the effluent through a flow cell in a Nuclear Chicago liquid
scintillation counter.

Autoradiograms were made of paper and thin-layer chromatograms, and
paper electrophoretograms using Kodak, Royal Blue, no screen, medical
X-ray film. Time of film exposure was 28 days.

Extraction and Fractionation Procedures

Initially, 807o ethanol was used as an extracting medium. However,
. early in this work it was found that water removed most of the radio-
activity from the tissues. Thus, water was used throughout the study.
Two extraction and fractionation procedures were primarily used: a)

28

dialysis-solvent fractionation, and b) column fractionation. Where
there are deviations from these procedures, they will be stated with
the results.
Method of Dialysis and Solvent Fractionation

V'Jater extracts were dialyzed using a cellulose acetate membrane
against water at 4° C. The radioactive components would pass readily
through the membrane. This initial clean-up procedure removed many
interfering substances and allowed the resulting solutions to be
manipulated through additional frac tionational procedures more quanti-
tatively, e.g., decreased the formation of emulsions. The dialyzable
portion was then partitioned between various organic solvents from an
aqueous solution which was either basic or acidic.
Column Frac tionational Procedures

Water extracts were first freed of most proteins and much of the
pigment material by precipitation with a small amount of 957o ethanol.

After settling, the precipitate was removed by centrifugation at 3980
X ^. The supernatant was then percolated through a 1 x 9 cm Dowex
50-X8 (100-200 mesh) ion exchange column in the hydrogen form. The
column was subsequently washed with water and 0.4N ammonium hydroxide
in 807o ethanol. Some of the resulting samples were further fractionated
on a Dowex 1-X8 column in the formate form.

A set of samples that had been dialyzed was passed through a water-
jacketed 135 X 0.64 cm column of polystyrene SO3H resin (type A). The
resin vas converted to the ammonium form by 4M ammonium hydroxide. The
column was operated at 66° C with a flow rate of 1.0 ml/min. The UV
absorbance at 290 m*/ of effluent from this column was monitored by a
Beckman DB spectrophotometer equipped with a Sargent SRL recorder.

29
A sample was also passed through a 35 g , 2 cm silicic acid column,
A step-wise elution sequence was used and 10 ml fractions were
collected and concentrated.

Paper and Thin-Layer Chromatography
All radioactive fractions separated were subjected to paper (22)
and thin-layer chromatographic techniques (123, 131). Specific •
parameters are given in the results section.

Paper Electrophoresis
A Spinco model R electrophoresis system was used at either pH
8.6 attained with B-2 Veronal buffer, 2.76 g diethyl barbituric acid,
15.40 g sodium diethyl barbituate (0.075 ionic strength) or pH 2.5
attained with 0 . 4N acetic acid.

RESULTS
Citrus Fruit Bioassay for Abscission

Using the citrus fruit bioassay, it was determined that the
maximum time required for uptake of lOAC for the most rapid rate of
abscission was approximately 24 hours (Table 1, Fig. 1). From these
results it- was assumed that whatever the fate of lOAC in the plant, a
24-hour period was an adequate time interval for the changes to occur.
Therefore, in all experiments where radioactive lOAC was applied to a
limited zone on the leaf surface, a 24-hour uptake period was used.

The citrus fruit bioassay was also used to screen additional
chemicals for their capacity to promote abscission (Table 2). Malonic
acid, 2 ,4-dinitrophenol , potassium iodide, diiodomethane , L-alanine,
cysteine, mannitol, sorbitol, glucono-delta-lactone , and ribose all
accelerated the rate of abscission of the explants. Sugar compounds,
other than the previous ones mentioned, either had no effect or delayed
abscission. Ascorbic acid strongly inhibited abscission (Table 2, Fig.
2) under the conditions of these tests.

Patterns of ^'^C02 Production from Tissues Treated with lOAC

Both carboxyl and methyl labeled lOAC-^'^C, when applied to a
limited zone on the surface of 'Pineapple' orange leaves, resulted
in a production of ^^C02 • This was the case for either leaves attached
to the plant (Fig. 3) or leaves detached from the plant and assayed in
a closed system (Fig. 4).
•'■^C02 Production from Attached Leaves

The^'^C02 production was periodically sampled at 1-hour intervals

30

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a.

'-'

X

X

iw

.

in

o

0)

4->

O

0)

r^

H

E

5

00

*r-*

^-^

W

U

c~

Cd

X

3

O

o

M

x:

O

i~i

dj

CU

OC

X

U-l

c

w

O

fl-

4_t

o

o

o

■<r

a

-

to

M-(

CJ

Pi

M-4

•— <

t5

W

a.

o

o.

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«

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o

r— i

c

oo

—

•H

[*<

o

o

ON

o

o

CO

NOISSIDSaV %0S ox S^iflOH

33

Table 2. Effect of various chemicals on the rate of abscission of
'Pineapple' orange explants.^

Molarity

Abscission

(7o)

Chemical

48

72

96

120

144

168Hrs.

Water control

0

10

50

100

lodoacetic acid

5

X

10-^

100

Mannose

5

.5

X

10-2

20

30

40

100

2

.8

X

10-2

0

0

20

100

Mannitol

5

2

.5
.8

X

X

10-2
10-2

100
100

Xylose

6,

.6

X

10-2

10

20

70

100

3

.3

X

10-2

10

20

70

100

Sorbitol

5,

.5

X

10-2

0

100

2,

.8

X

10-2

0

70

100

Galactose

5,

.5

X

10-2

10

10

50

70

100

1.

.8

X

10-2

0

0

40

70

100

Glucono-delta-

5,

.5

X

10-2

30

100

lactone

2.

.8

X

10-2

20

90

100

Fructose

5,

.5

X

10-2

0

20

60

80

100

2,

.8

X

10-2

0

30

80

100

Ribose

6,

,6

X

10-2

70

80

80

100

3,

.3

X

10-2

80

100

Dextrose

5,

.5

X

10-2

0

0

-^0

40

100

2,

,8

X

10-2

0

0

30

50

90

100

Ethyl iodide

10-3

5

10

10

45

85

90

5

X

10-^

5

20

35

55

75

80

10-^

0

5

5

15

75

100

Methyl iodide

10-3

25

55

55

55

55

70

5

X

10-4

10

30

30

30

30

40

10-4

0

5

10

15

15

15

Potassium

10-3

80

100

iodide

5

X

10-4

30

70

85

100

10-4

5

25

75

75

100

Diiodomethane

10-3

70

100

Malonic acid

10-3

75

80

100

Cysteine

10-3

10

70

100

Cysteine -:-

10-3

iodoacetic

-I-

70

100

acid

5

X

10-4

2,4-dinitrophenol

10-3

50

75

100

2 ,4-dinitrophenol

10-3

-f- iodoacetic

+

65

100

acid

5

X

10-4

L-alanine

10-3

55

100

L-ascorbic acid

5

X

10-4

15

15

15

15

20

30

^Oranges harvested in January, 1967,

34

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01

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u

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t— 1

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37

for 24 hours in one test and 48 hours in another test (Fig. 3). The
release of ^'^C02 still was increasing over the longer time period.
The initial peak at the 1-hour sample (Fig. 3, Exp. II) was variable
and could be the result of trapping any initially volatile components
as lOAC is decomposed somewhat by light. However, the level should
have been maintained if chat were the case. The peak was obtained
whether or not the plexiglass chamber was darkened.
^^C02 Production from Detached Leaves

The 1^C02 production from detached leaves in a closed system

indicated that a small fraction of both IOAC-1-l^C and IOAC-2-l^C was

metabolized to ■'■'^C02 (Fig. 4). Slopes of the •'■^002 production curves

show that both the methyl and carboxyl lOAC are metabolized at similar

rates .

Effect of Time of Extraction on the Amount of Radioactivity
Found in the Extract and Remaining in the Tissues

Treatment of 'Pineapple' (Table 3) and 'Valencia' (Table 4) orange
leaves with IOAC-l--'-^C showed that in both varieties the amount of
radioactivity extracted with ethanol did not diminish over 48 hours.
This indicated that the lOAC was not metabolized to cell wall material
or any macromolecules that were not soluble in ethanol. It also demon-
strated that a large portion of the lOAC was not lost as l'^C02 .

The amount of radioactivity remaining in the 'Pineapple' residue
was a very small fraction of the total radioactivity and, again, the
amounts were not significantly different with time (Table 5). The amount
of radioactivity in the 'Valencia' residue was also a very small portion
of the total (Table 6). However, there was a slight drop in the radio-
activity remaining in the residue over the 48-hour time interval. This
again indicated that there was very little metabolism of lOAC to macro-
molecules or cell wall material.

38

Table 3. Ethanol extractable radioactivity in 'Pineapple' orange leaf
disks treated in 2 ml of 5 x 10"^M IOAC-1-l'^C for 3 hours?

Radioactivity in ethanol extract (cpm)^
Hours Exp. I II III IV y_

1 11,520 12,350

6 9,730 10,120

10 11,140 12,000

24 12,030 10,520

48 11,300 10,010

^Leaf disks were extracted with 80% ethanol after 1-, 6; 10-; 24- and 48-
hour intervals following treatment. The specific activity of the
IOAC-1-l^C was 1.4 mc/m mole.

t'F= <..01 N. S. with respect to time of extraction.

11,130

8,570

7,480

8,660

6,110

8,470

8,260

8,510

9,070

9,060

9,980

8,040

9,180

8,700

7,970

39

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40

Table 5. Radioactivity remaining after ethanol extraction in

'Pineapple' orange leaf disks after treatment in 2 ml of 5 x lO'^M
IOAC-1-^'^C for 3 hours.^

Radioactivity in residue (cpm/mg fresh weight)"

Hours Exp. I II III IV y_

I 2.6

6 1.8

10 3.5

24 2.1

48 1.7

^Leaf disks were extracted with 807„ ethanol after 1-, 6-, 10-, 24-, and 48-
hour intervals following treatment. The specific activity of the
IOAC-1-l'^C was 1.4 mc/m mole.

°F= <, 1 N. S. with respect to time of extraction.

3.0

1.5

1.4

2.0

3.0

1.3

1.9

1.6

2.6

1.9

1.5

1.0

1.3

1.9

0.4

1.8

2.5

0.8

1.6

0.7

41

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42

Metabolism of lOAC Applied to Leaves in Drops
Both 'Pineapple' and 'Valencia' orange varieties metabolized
labeled IOAC-1-l^C. Approximately 60 to 80% of the total radioactivity
absorbed by the leaves could be extracted with water and was dialyz.able
(Tables 7 and 8). The average value for the dialyzed components for
the 'Pineapple' variety was 11.9% and that for the 'Valencia' variety
was 63.77o. Since most of the radioactive components would pass through
a membrane, and since this procedure removed interfering substances
that hindered solvent partitioning, this technique was used prior to
other fractional procedures.

Solvent partitioning of the dialyzable portion of the radioactivity
indicated that less than 107o of the radioactivity in an acidic water
extract was soluble in ether (called the ether fraction) (Tables 7
and 8). The aqueous portion from this separation was termed the water
fraction and the large percentage of radioactivity remaining in this
fraction indicated that the labeled metabolites formed from the lOAC-
l^C were polar. This was substantiated by showing that various organic
solvents did not partition any radioactivity from this water fraction
(Table 9).
Measurement of the Free lOAC in the Tissues After a 24-Hour Uptake Period

Free IOAC-l--'-^C in the extracts from orange leaves was removed from
the dialyzed water extract by partitioning between aqueous HCl (pH 3.0)
and diethyl ether. Only lOAC was detected in the ether fraction (Fig. 5
and Table 10 for Rf values of paper chromatography separation) and no
further separation was done on this fraction. Both 'Pineapple' and
'Valencia' leaves, after the 24-hour time period, had approximately the
same (less than 107o of activity remaining in the leaf) percent of free
IOAC-1-l^C in the partitioned diethyl ether fraction (Tables 7 and 8).

43

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Table 9. Distribution of radioactivity in fractions of a water extract
of 'Pineapple' orange leaves treated with IOAC-1-l'^C?

Fraction

Total

% of

cpra

initial value

Basic

Solution

Water extract"

63,700

-

pH 8.0

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0

-

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0

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0

-

Ether

0

-

Acidic

Solution

EtherC

2,460

3.7

pH 3.0

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0

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0

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Benzene

0

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Toluene

0

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Water

67,164

105

^The lOAC was applied in 2 , 10 ^1 drops, 1 on each side of the main
vein. Time of uptake of lOAC was 24 hours. The water extract was
made basic (pH 8.0) with NaHC03, then adjusted to pH 3.0 with 2N HCl,

Dialyzable portion of extract.

'^Extracted with diethyl ether (10 ml, 5 times).

46

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0

ETHER ACID FRACTION

IOAC-1-l^C IOAC-2-l^C

Fig. 5. Autoradiogram of thin-layer separated ether-acidic-
par titioned-frac tion of 'Pineapple' orange leaves treated
with IOAC-1-l'^C. The lOAC was applied in 2, 10 j» 1 drops,
1 on each side of the main vein. Developed in butanol:
ethanol rwater , 65:10:10, v/v/v, on Eastman chromagram
silica gel sheets (type K 301R2).

47

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48

'Pineapple' leaves treated with IOAC-2-l^C also had approximately the
same percentage of free lOAC in the ether fraction (Table 11). These
results indicated that most of the lOAC inside the tissue had been
metabolized by both 'Pineapple' and 'Valencia' leaves to a metabolite
or metabolites that were extremely polar in nature since the major part
of the activity was retained in the water fraction. There was little
radioactivity remaining in the leaf residue material. Since both the
methyl and carboxyl labeled lOAC resulted in the same ^'^C distribution
pattern, it appeared that the 2 carbons were metabolized similarly.
Carboxyl and Methyl ^■'^C-labeled lOAC Metabolites

When carboxyl or methyl I'^C-labeled lOAC was applied to 'Pineapple'
and 'Valencia' orange leaves, both metabolized approximately 90% of the
absorbed lOAC to the same metabolites. Paper chromatograms of the
water fraction contained 3 distinct metabolites of which 2 appeared
to be positive to ninhydrin (Fig. 6). Referring again to Table 10,
paper chromatography of the water fraction in 6 solvent systems showed
that 'Pineapple' leaves treated with either IOAC-1-^'^C or IOAC-2-l^C
formed the same metabolites based on Rf values. When separated on a
paper electrophoretic system, these same compounds in the water phase
were shown to be amphoteric (Fig. 7). At least 3 major metabolites
and several minor ones were shown by electrophoresis to be formed by
both 'Pineapple' and 'Valencia' leaves from IOAC-1-l^C and IOAC-2-l^C
(Figs. 8-11). lOAC moves towards the anode under these same conditions
(Fig. 12). These tests indicated that at least 3 major metabolites
were formed in both 'Pineapple' and 'Valencia' leaves when treated
with either IOAC-1-l'^C or I0AC-2-l'+C. One of the metabolites (III)
appeared in greater concentration during the second day of dialysis
(Fig. 13). This probably indicated bacterial contamination of the
system since this compound (III) accumulated .

49

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'PINEAPPLE'

IGAC-l-l-^C

'VALENCIA'

Fig. 6. Autoradiograms of paper chromatographic separation
of l^C-metabolices from 'Pineapple' and 'Valencia' orange
leaves treated with IOAC-1-l^C. The lOAC was applied in
2, 10 yl drops, 1 on each side of the main vein. Time
of uptake of lOAC was 24 hours. Chroma togramed on Whatman
#3 paper and developed in N-propanol :water , 6:4, v/v. The
letters and lines designate labeled metabolites that were
positive to ninhydrin. Color code: P=purple.

51

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52

ANODE

ORIGIN

CATHODE

'VALENCIA'

'PINEAPPLE'

'PINEAPPLE'

'PINEAPPLE'

Fig. 8. Autoradiogram of l^C-metabolites from 'Pineapple'
and 'Valencia' orange leaves treated with IOAC-1-l'^C. The
lOAC was applied in 2 , 10 ^1 drops, 1 on each side of the
main vein. Time of uptake of lOAC was 24 hours. Separated
by paper electrophoresis in 0.4N acetic acid buffer, pH 2.5
for 4 hours at 500 volts.

53

500

CATHODE

Fig. 9

ORIGIN

ANODE

Electrophoretic separation of l^C-metabolites resulting
from drop application of IOAC-1-l^C to 'Pineapple' orange leaves.
Developed for 4 hours at 500 volts in 0.4N acetic acid buffer
pH 2.5. '

54

300

CATHODE

ORIGIN

ANODE

Fig. 10. Elec trophoretic separation of •'-^C-metabolites resulting
from drop application of IOAC-1-l^C to 'Valencia' orange leaves,
Developed for A hours at 500 volts in 0.4N acetic acid buffer,
pH 2.5.

55

300

o

CATHODE

ORIGIN

ANNODE

Fig. 11. Electrophoretic separation of ■'■^C-metabolites resulting
from drop application of IOAC-2-l^C to 'Pineapple' orange leaves.
Developed for 4 hours at 500 volts in 0.4N acetic acid buffer,
pH 2.5.

56

300

CATHODE

ORIGIN

ANODE

Fig. 12. Electrophoretic pattern of 5 x lO^^M lOAC-l-^'^C.

Developed for 4 hours at 500 volts in 0.4N acetic acid buffer,
pH 2.5.

57

300

CATHODE

ORIGIN

MODE

Fig. 13. Electrophoretic separation of l^C-metabolites resulting
from drop application of IOAC-1-l'^C to 'Pineapple' orange leaves.
Metabolites appearing during 24-48 hours' dialysis time. Developed
for 4 hours at 500 volts in 0.4N acetic acid buffer, pH 2.5.

58

Initially, an attempt was made to determine whether or not the
metabolites would be formed in macerated tissue. Also, tests were
conducted to see whether the compounds could be formed either in the
supernatant or the residue from macerated leaves. One test was
conducted in a phosphate buffer (pH 6.2) system, several in water,
and 1 in ethanol. In 1 test metabolites were formed from lOAC .
However, in 5 subsequent tests, including the phosphate buffer experi-
ment, no labeled metabolites were formed. This indicated that macerating
the orange tissues destroyed the capacity to metabolize lOAC . In the 1
test where metabolites were found, some islands of intact living cells
might have remained after the maceration.

A sample extracted from 'Pineapple' leaves treated with lOAC-l-^'^C
was subjected to column chromatography on silicic acid in a system
designed to separate flavonoid and phenolic compounds. The result was
that the labeled metabolites were not readily soluble in ethyl acetate
or methanol. The major part of the radioactivity appeared in the first
few 10 ml fractions of a methanol :water , (v/v) eluting solvent. Thus,
the compounds that were labeled were polar in nature. Also, furcher
separations by thin-layer chromatography of these radioactive fractions
indicated that ^^C labeled sugars, flavonoid or phenolic compounds (Fig.
14) were not formed as a result of the metabolism of lOAC . Paper
chromatography of 2 of the most radioactive fractions showed the presence
of 3 labeled compounds, 2 of which reacted to ninhydrin. These 2
corresponded in Rf values to glutamic and aspartic acids (Table 12).
This indicated that at least the carboxyl carbon of lOAC is metabolized
to amino acids. Electrophoretic separation of the fractions with the
highest amount of radioactivity after elution from the silicic acid
column showed that only 2 of the 3 major metabolites had been recovered.

Fig. 14. Autorad iogram of polyamide thin-layer sheet separation
of l^c-nie tabolites formed by 'Pineapple' orange leaves treated
with IOAC-1-l'^C. A sample of the water extract was fractionated
on a silicic acid column. These radioactive fractions,
appearing at the start of a methanol :water , v/v elution sequence,
were developed on a polyamide thin-layer sheet 3 times in
methanol :nitromethane , 2:5, v/v.

The circles depict the compounds visualized with
ethanolic aluminum chloride. Tentative identification is
given for some of the non-labeled metabolites. Code: P=prunin;
N=naringin; R=rhoitolin.

Fraction

1-

111-

•114

2-

115

3-

116-

■119

4-

120

5-

121-

■124

6-

125

7- 126-129 13- 143-145

8- 130 14- 146-147

9- 131-134 15- 148-149
10- 135 16- 181-182

"ll- 140 17- Naringin

12- 141-142

60

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61

Table 12. Rf values from paper chromatograms of labeled metabolites
formed by 'Pineapple' orange leaves treated with IOAC-l--'-^C.^

Rf values of labeled metabolites
Solvent systems"

2

.17, .31
,05, .12, .31
.17

.33

The lOAC was applied in 2 , 10 yl drops, 1 on each side of the main
vein. Time of uptake of lOAC was 24 hours. Fractions were eluted
from a silicic acid column with methanol rwater, v/v, and spotted on
Whatman #1.

1 - Butanol :water racetic acid, 4:5:1, v/v/v (upper phase).

2 - Methanol :water :acetic acid, 19:1:1, v/v/v.

3 - Phenol:water, 100:20, v/v in 0.047, 8-hydroxyquinoline.

Fraction

115

.1,

.20

.12,

.32

Fraction

121-124

.17

.10,

.31

Aspartic

acid

.16-

.22

.14

Glutamic

acid

.22-

.28

.27

62
The other metabolite could have been present in some other fraction
or it could have remained on the column. The latter was shown to be
more likely because subsequent tests showed that if the extract was
dried in the presence of silicic acid, then most of the radioactivity
was irreversibly bound to the silicic acid, i.e., could not be removed
by water.

'Pineapple' and 'Valencia' orange leaves formed the same metabolites
when treated with IOAC-1-l^C. However, the amount of radioactivity in
'Pineapple' leaves treated the same as 'Valencia' leaves was approx-
imately 3 times greater (Tables 7 and 8). This would seem to indicate
that lOAC was either more readily absorbed into 'Pineapple' leaves
than it was into 'Valencia' leaves, or that the resulting metabolites
were less readily translocated out of 'Pineapple' leaves.
Acetate Metabolism by Orange Leaves

'Pineapple' orange leaves treated with acetate-1-l^C in the same
manner as those treated with lOAC-^'^C resulted in a slightly different
pattern of metabolites. Less radioactivity remained inside the
dialysis membrane with acetate treated leaves (Table 11) than was
present in lOAC treated leaves. A greater percentage of the radio-
activity extracted by water was partitioned into the acidic ether
fraction, indicating either more acetate present or a larger percentage
of acidic materials labeled. There was little radioactivity remaining
in the leaf residue material, indicating little production of non-water
soluble materials. The amount of radioactivity absorbed, however, was
not enough to permit separation by either paper chromatography or
paper electrophoresis. For this reason, the petiole-absorption
technique was used as it resulted in a much greater uptake of the
acetate-1-l^C.

63
Metabolism of lOAC Applied to the Leaves by Petiole Uptake
Amount of Free lOAC Remaining la the Tissues

The amount of unme tabolized lOAC remaining in the 'Pineapple' and
'Valencia' leaves after uptake for 2 hours was found to be not greater
than 307o of the total extractable radioactivity. This was calculated
from the total radioactivity in the acidic and neutral frac tions (water
fraction) from a Dowex 50-X8 column (Table 13). Subsequent separation
of this fraction by paper chromatography showed that at least 1 other
metabolite was present in both varieties (Figs. 15 and 16). The results
indicated that the free lOAC (Rf .72-. 74) represented a small fraction
of the 307o radioactivity in the acidic and neutral fraction. This
compared favorably with the approximate 57o free lOAC value obtained
in the drop application experiments.

Further fractionation of the water fraction (from the Dowex 50-X8

column) on a Dowex 1-X8 column (formate) with subsequent thin-layer

separation on Eastman chromagram silica gel films, showed that labeled

compounds appeared in the water washes and in the 17o formic acid elution

(Fig. 17, numbers 1 and 3). Since these compounds were initially eluted

from a Dowex 50-X8 column in the water wash, they were probably either

neutral or acidic in nature.

Metabolites Formed as a Result of Petiole Uptake of Carboxyl and Methyl-
Labeled lOAC

'Pineapple' and 'Valencia' orange leaves both metabolized either

IOAC-l--'-^C or IOAC-2-l^C to the same compounds found in either the water

or the ammonium fractions eluted from the Dowex 50-X8 column (water

fraction metabolites discussed previously). The Rf values of the various

metabolites determined from paper chromatograms in 3 solvent systems

clearly show that in all cases the same metabolites were formed (Tables

64

Table 13. Distribution of radioactivity in ethanolic ammonium and
water fractions collected from a Dowex 50-X8 columnf .

Percent radioactivity in fractions

Sample

Exp. I

II

Water

Ammonium

Water

Ammonium

86

14

80

20

83

17

89

11

59

41

23

77

16

84

29

71

21

79

33

67

33

67

16

84

98

2

98

2

'Pineapple' acetate-1-l'^C
'Valencia' acetate-1-l^C
'Pineapple' IOAC-1-l'^C
'Valencia' IOAC-1-l^C
'Pineapple' lOAC-Z-l-^C
'Valencia' lOAC-Z-l'^C
'Pineapple' 131ioAC

^Extracts of 'Pineapple' and 'Valencia' orange leaves in which either
IOAC-1-l'^C, IOAC-2-l^C, 131I0AC, or acetate-1-l'^C were applied by
petiole uptake. Uptake time was 1 hour, after which the leaves were
placed in water for an additional hour.

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Fig. 17. l^C-metabolites from 'Pineapple' orange leaves

created with either IOAC-1-l'^C or acetate-1-l'^C. The radio-
active compounds were applied to the leaves by petiole uptake.
Time of absorption was 1 hour, after which the leaves were
transferred to water for an additional hour. The leaf extracts
were eluted from a Dowex 1-X8 column (formate) and separated on
Eastman chromagram silica gel thin-layer films. Developing
solvent was butanol :water :acetic acid, 4:5:1, v/v/v (upper
phase) .

The circles depict compounds positive to bromcresol

green.

Fraction

1 IOAC-1-l'^C, water fraction,

2 acetate-1-l'^C , water fraction,

3 IOAC-1-l'^C, water fraction,

4 acetate-1-l'^C , water fraction,

5 IOAC-1-l'^C, water fraction,

6 acetate-1-l'^C , water fraction,

7 IOAC-1-l^C, water fraction,

8 acetate-1-l'^C , water fraction,

2, 3, 4, water washes.
2, 3, 4, water washes.
17o formic acid elution.
1% formic acid elution.
57o formic acid elution.
57o formic acid elution,
407o formic acid elution.
407o formic acid elution.

68

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69
14, 15, and 16). A comparison of these Rf values with standard Rf
values indicated that at least 2 of the components in every solvent
system correspond to glutamic and aspartic acids. These unknown
compounds were positive to ntnhydrln. Thus, they were tentatively
identified as being glutamic and aspartic acids. The other metabolites
were not identified.

A scan of the ammonium eluted fraction from a polystyrene column
shows the presence of 6 labeled metabolites in 'Pineapple' orange
leaves (Fig. 18). This is in agreement with the results obtained by
paper chromatography in a butanol solvent system in which there were
also 6 metabolites. However, the fractions from the polystyrene column
were monitored for radioactivity but not collected; therefore, no Rf
values from other forms of chromatography were obtained.

The 'Pineapple' water and ammonium fractions from the hydrogen
column, separately, were also eluted from a formate column. These
fractions from the formate column were further separated by paper
chromatography using 3 solvent systems. Again, in the ammonium fraction
sample. from the Dowex 50-X8 column (fractionated on the formate column),
2 of the spots reacted to ninhydrin and these had the same Rf values as
glutamic and aspartic. In fact, the "fit" of the compounds eluted from
the formate column was better than that from the Dowex 50-X8 column
ammonium eluted compounds, probably because the extract contained fewer
components (Rf values - Tables 17, 18, and 19). These same fractions
from the Dowex 1-X8 column (formate) were also separated by thin-layer
chromatography using Eastman chromagram thin-layer chromatographic films.
Again, 2 radioactive, ninhydrin positive spots, corresponding to glutamic
and aspartic acids, were separated from the VA formic acid elution (Fig.
19, number 3). These components also gave the correct ninhydrin colored

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^

Fig. 19. l^C-metaboli tes from 'Pineapple' orange leaves treated
with either lOAC-l-^^C or ace tate- 1-l^C . The radioactive com-
pounds were applied to the leaves by petiole uptake. Time of
uptake was 1 hour, after which the leaves were placed in water
for an additional hour. The leaf extracts were eluted from a
Dowex 1-X8 column (formate) and separated on Eastman chromagram
silica gel sheets. Developing solution was butanol ;water :
acetic acid, 4:5:1, v/v/v (upper phase).

The circles designate metabolites that were positive
to ninhydrin (0.25% in acetone). Color code: P=purple, B=blue.

Fraction

1 IOAC-1-l'^C,

2 acetate-1-l'^C,

3 IOAC-1-l^C,

4 acetate-1-l'^C,

5 IOAC-1-l^C,

6 IOAC-1-l^C,

7 glutamic acid.

8 aspartic acid.

9 alanine.

1st water wash.
1st water wash,
ammonium fraction,
ammonium fraction,
ammonium fraction,
ammoaium fraction.

1% formic acid elution.
17o formic acid elution.
57o formic acid elution.
2, 3, 4 water washes.

78

0.9 r

79
chromaphore for the 2 amino acids (blue for aspartic , and purple for
glutamic). There were 2 other metabolites in this elution fraction
that did not react to ninhydrin, but 1 of them corresponded closely to
ninhydrin posi tive (Fig . 19, number 2) compound obtained from the
metabolism of acetate by 'Pineapple' leaves. Two metabolites also
were present in the 57o formic acid wash (Fig. 19, number 5) and these
same 2 were in the combined water wash (Fig. 19, number 6). These
did not react to ninhydrin even though 1 had an R^ similar to aspartic
acid. However, since it failed to react with ninhydrin and since it
was eluted from the column later than aspartic acid, it would seem that
it was a different compound.
Metabolites Formed as a Result of Acetate-1-^^C Metabolism

Within the 2-hour uptake time interval of the experiment all of the
acetate in the 'Pineapple' leaves had been metabolized, since subsequent
separation of the extraction solution failed to detect any free acetate.
However, approximately 757o of I'^C-acetate either was exchanged or was
volatile in the solvent systems used. Thus, if any acetate was
unmetabolized , it would probably be lost in the chromatography systems.
Approximately 807o of the extracted radioactivity came through in the water
wash of the Dowex 50-X8 column, indicating that the majority of the
radioactivity was in acidic (if not acetate) or neutral compounds (Table
13). This was almost an exact reverse of the IOAC-1'^C metabolites since
most of these were in the ammonium fraction. No substantial radioactivity
remained behind in the leaf residue material. At least 2 metabolites
were present in the water fraction (Table 17). There were also at least
3 (Table 17) labeled metabolites in the ammonium fraction from the
Dowex 50-X8 column which had been, in turn, fractionated on a formate
column. Two of these metabolites (in the ammonium fraction) were ninhydrin

80
positive and corresponded to glutamic and aspartic acids, as did the
lOAC-^'^C metabolites.

Thin-layer chromatography of the ammonium fraction (from the DoweK
50-X8 column) further eluted from a formate column, showed that 2
ninhydrin-positive , radioactive metabolites were present in the VL
formic acid eluttng fraction (Fig. 19, number 4) with similar Rf values
to those of glutamic and aspartic acids. The first water wash from the
formate column also removed a radioactive, ninhydrin-positive metabolite
(Fig. 19, number 2). However, the Rf value was^^ 0.36 and the closest
amino acid was alanine at 0.40. The crude extract had previously been
checked for phenolic amine activity. Since no phenolic amines were
labeled, this metabolite with an Rf of 0.36 could possibly be alanine.
Thin-layer chromatography of the water fraction (from the Dowex 50-X8
column) further eluted from a formate column showed that at least 1
metabolite was present in the 57o formic acid elution and that this
metabolite was probably acidic since it formed a yellow chromaphore with
bromcresol green (Fig. 17, number 6). Another metabolite with almost
the same Rf,but appearing in the 407o formic acid wash, was also acidic
(Fig. 17, number 8).

Separation of the metabolites on a polystyrene column showed that
there were 4 metabolites in the ammonium fraction from this column
(Fig. 20). Two components (peaks c and h) were common to both lOAC
metabolism and acetate metabolism (compare Figs. 18 and 20 with Fig. 21
which was a scan of a composite sample). Therefore, since the metabolism
of lOAC produced some compounds similar to those formed when acetate was
metabolized, it would appear that some of the lOAC was metabolized as
acetate. However, it was clear that there was a difference between the
metabolism of lOAC and that of acetate in 'Pineapple' leaves.

81

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83

Metabolism of 131iOAC

^-"^lOAC treatment of 'Pineapple' leaves resulted in a compound
with a different Rf value that any formed in the lOAC-l^'C metabolism
(compare Table 20 to Tables 14, 15, and 16). This metabolite was not
positive to ninhydrin. Almost all of the activity was present in the
water fraction from the Dowex 50-X8 column and none in the ammonium
fraction (Table 13).
Metabolism of Nal31l

Sodium 131x treated 'Pineapple' leaves resulted in a metabolite
with an Rf value similar to the 131xoAC compound (Table 20). Approximately
307o of the activity was lost from the solution when the proteins and
green pigments were precipitated. The remainder of the activity came
through the Dowex 50-X8 column in the water fraction (Table 21).

84

Table 20. R£ values from paper chromatograms of metabolites formed

by 'Pineapple' orange leaves treated with either l^lioAC or
NalSlia

Sample

Rf values of labeled metabolites
Solvent systems"
1 2 3

131

lOAC

'Pineapple' l^lxoAC water
fraction

'Pineapple' Na^^lj water
fraction

.85

.34-. 38

.32-. 34

,70-. 72
.60

.58

.64
.26

.23

^The radioactive compounds were applied to the leaves by petiole uptake,
Time of uptake was 1 hour, after which the leaves were transferred to
water for an additional hour. The samples were fractionated from a
Dowex 50-X8 column. They were then spotted on Whatman #1.

1 - Butanol rwater :acetic acid, 4:5:1, v/v/v (upper phase).

2 - Methanol :water :acetic acid, 19:1:1, v/v/v.

3 - Phenol :water, 100:20, v/v, in 0.04% 8-hydroxyquinoline.

85

Table 21. Distribution of radioactivity in fractions from 'Pineapple'
orange leaves treated with sodium l^l^odide and eluted from a Dowex
50-X8 column?

Fraction

7o of total radioactivity

Ethanol precipitated

Water

Ammonium

34

63

3

^The radioactive compound was applied to the leaves by petiole uptake.
Time of uptake was 1 hour, after which the leaves were transferred to

water for an additional hour,

DISCUSSION

Both 'Pineapple' and 'Valencia' orange tissues will metabolize
lOAC . In both instances, the patterns of labeled metabolites formed
from lOAC-^'^C were the same. This was the case whether the lOAC was taken
into the leaf through the surface or through the petiole. There was a
greater quantity of IOAC--'-'^C absorbed into the tissues through the
petiole than through the surface of the leaf. With the petiole uptake
method, there was a concomitant increase in the quantity and the
number of labeled metabolites. This increase in quantity and number
found could be a consequence of the greater uptake. However, it might
possibly be that the metabolism of lOAC in the petiole uptake tests
was different in some respects simply because of the route of entry of
the lOAC.

The reactions occurred over a relatively short period of time
since only a very small percentage of the total radioactivity in the
leaves remained as lOAC after only 2 hours. Also, the same results,
namely, only a small percentage of free lOAC , were found with the surface
application experiments. The duration of this test was 24 hours.

There was no significant incorporation of ■'-^C into cell wall
materials, macromolecules , sugar compounds, or fats and it appeared
that there were no phenolic compounds labeled. However, most of these
determinations were made on extracts from leaves treated by surface
application of O.IM lOAC. Absorption rates of lOAC appeared to be
slow under these conditions and not as much lOAC entered the leaves
as with the petiole uptake tests. Since more labeled metabolites were

86

87
found in the latter case, some of these unknowns might be either a
sugar or a phenolic compound. No fats, proteins, or cellular debris
left after ethanolic or water extractions were labeled in either case.

Two of the labeled compounds formed In the leaves were identified
as glutamic and aspartic acids. This was based on similarities of Rf
values and ninhydrin color reactions on chromatograms developed in 3
different solvent systems. These compounds were also retained on a
Dowex 50-X8 column and appeared in the 17, formic acid elution of a
Dowex 1-X8 column (formate). Both facts indicated that the molecules
were ionizable and could be amino acids.

These 2 amino acids were probably 2 of the major metabolites
isolated from the leaf-surface application experiments. Electro-
phoresis showed that these were charged and compounds I and II reacted
to ninhydrin. Also, paper chromatograms of these extracts showed 2
ninhydrin-positive, labeled metabolites were present with Rf ' s similar
to glutamate and aspartate.

Some of the unknowns were also bound to a Dowex 50-X8 column and
eluted from a Dowex 1-X8 column (formate) with water or with various
concentrations of formic acid. These were then organic cations but
their exact nature was unknown. Possibly there were other labeled
amino acids that were below the limit of detection by ninhydrin. Also,
both methyl and carboxyl l^C labeled lOAC treated orange leaves produced
■'■^002 in approximately the same patterns. This indicated that acetate
was being metabolized and the most likely pathway would be through
organic acids then to amino acids. Therefore, labeled organic and
amino acids would seem to be the logical components to be produced.
Glutamate and aspartate pools in the orange leaf are fairly large (148),
which would lend support to this idea.

88

The results indicated that some of the lOAC was metabolized
similarly to acetate. Labeled acetate applied to orange leaves resulted
in at least 2 to 3 of the same labeled compounds as did lOAC . Again,
labeled glutamic and aspartic acids were present in both cases. Also,
labeled alanine was tentatively identified as a component arising from
acetate-1-l^C metabolism.

Other labeled compounds appeared with lOAC that were not present
with acetate. A portion of the lOAC could have been metabolized
differently, or could have affected metabolism, per se. However, it
was clear from the CO2 pattern and the labeled metabolite patterns of
acetate and lOAC that a large portion of the lOAC was broken at the
I-C bond.

The metabolic patterns of l^llOAC and lOAC-^^C indicated further
that this disruption occurred in at least 90 to 957o of the lOAC in the
tissue. The time of bond separation was not readily apparent. However,
light was not the agent that catalyzed this reaction. Tests of lOAC-^^C
applied to a cellulose pad and kept in an acidic medium showed that no
discernible radioactivity was lost from that surface during sunlighted
or darkened exposures. Acetic acid would have been volatile under
these conditions. lOAC does decompose in light but the rate is
apparently very slow. It has been calculated to be 0.057o/hour (100).
Therefore, the I-C bond was being reacted upon by some other agent.

For glutamic and aspartic acids to form, it would seem logical
that the I-C bond would have to be disrupted before the acetate was
metabolized. This idea was supported by the capacity of the orange
leaves to form the same iodinated compound from either ■'■-'■'■lOAC or
sodium 1-^liodide. The identity of this compound was unknown but it
was not free ■'•-^•'■iodine. The component was not retained on a Dowex

89
50-X8 column. About 307, of the radioactivity with l^^IOAC treated
leaves was precipitated by alcohol. This could indicate that the
■'•■^•'•iodine was bound to a large molecule, probably protein. Since
potassium iodide will promote orange explant abscission, it appears
that the iodide portion of lOAC could be responsible for a portion of
the abscission-accelerating activity. The lOAC may act as a better
carrier for iodine since potassium iodide in a spray will not promote
orange abscission but lOAC will. The iodide ion does not move readily
into the leaf.

From previous work (76, 77), it has been shown that abscission of
fruits of 'Pineapple' and 'Valencia' orange varieties are different
when subjected to the same lOAC sprays. Yet, the data presented here
show that there were no differences in the labeled metabolites formed
from 'Pineapple' or 'Valencia' orange leaves treated with lOAC . However,
there was approximately 3 to 4 times the amount of radioactivity in the
'Pineapple' leaves as that of the 'Valencia' leaves from the surface
application experiments. This indicated either a greater uptake of
the lOAC by 'Pineapple' leaves or a slower rate of translocation. A
previous investigation on lOAC uptake (152) had shown a similar pattern
and appearance of radioactivity in both varieties with time. It was
concluded that there was no difference in translocation of labeled
materials. From the present data on uptake of lOAC from surface
applications vs petiole uptake, it would appear that the difference
between 'Pineapple' and 'Valencia' in the abscission response was due
to a difference in surface uptake.

Cross-sections of the leaves from both orange varieties were
examined and no difference in the cuticle thickness was observed.
In fact, the 'Pineapple' cuticle, though variable, appeared to be

90
slightly thicker. Average values of a relative scale used to measure
cuticle thickness were: 'Pineapple', 2.736; and 'Valencia', 2.464.
Therefore, cuticle thickness cannot account for the observed differences
in radioactivity in the leaves or in the differences with respect to
abscission.

'Valencia' leaves also differed from 'Pineapple' leaves In the
amounts of radioactivity in the residue. The amount of radioactivity
in the 'Valencia' leaf residue decreased with time. This indicated
that there was no significant synthesis of l^C from lOAC to macro-
molecules occurring during this time. The reason for the difference
in the radioactivity remaining in the residues after extraction between
the 2 varieties is not known. However, the radioactivity in the residue
was a small portion of the total radioactivity (< 0.1%).

Unfortunately, these metabolic studies did not indicate the mode
of action of lOAC on orange fruit abscission. The lOAC was metabolized
very quickly in the tissues; therefore, it would seem that the action
on abscission, per £e,was not entirely through the action of intact
lOAC as an inhibitor of metabolism. However, both malonic acid and
2 ,4-dinitrophenol promoted orange abscission and these are both metabolic
inhibitors. Also, it has been suggested that lOAC acts as an uncoupler
of high-energy phosphate systems (47). Dinitrophenol acts similarly
(46); therefore, the effect of some of the lOAC might be on high-energy
transfer systems. Also, there could be an additive effect of the iodine
and the unmetabolized lOAC.

Additions of sugars have been shown to inhibit abscission in many
systems (13, 15, 27, 96, 108), including the orange explant test (168,
169). This study yielded the same results, namely, that most sugars
delayed abscission. Ascorbic acid was found to delay the rate of

91
abscission of the orange explants. Other workers have found that
ascorbic acid enhanced orange fruit abscission (48). Their test
system was different from the orange explant test and the concentration
tested in their investigation always exceeded lO'^M.

lodoacetic acid has also been shown to change the metabolic
pathway of glucose from glycolysis to the pentose shunt (55, 90).
Ribose slightly accelerated the rate of abscission of the orange
explants, so this could suggest an involvement of the pentose shunt
with abscission.

Certain amino acids have been shown to affect abscission. Alanine
enhanced the rate of abscission of the orange explants as it does bean
explants (41, 136, 137, 140). However, the effect of alanine in the
bean explant bioassay was found to be related to ethylene production
(137), but this has not been ascertained for citrus. Cysteine, also,
slightly promoted orange fruit abscission. In conjunction with lOAC
only a slight reduction in the abscission rate produced by lOAC alone
was found. If lOAC acted as a sulfhydryl enzyme inhibitor, possibly
flooding the tissues with sulfhydryl material might have reduced the
effect of lOAC . There was no interaction between cysteine and lOAC on
citrus fruit abscission.

Acetic acid applied as a spray on whole trees or in the explant
test did not promote abscission. Therefore, the products of
metabolism of the acetate portion of lOAC probably are not the active
agents in initiating the events leading to abscission. Since some
products from metabolism of lOAC were not found with acetate- treated
leaves, these might be important. However, probably more important
is the iodine compound or the fact that the iodine ion might exist.
Potassium iodide will promote orange abscission in the explant test,

92
but not when sprayed on trees (76). This suggests that the iodine
was active in promoting abscission and that lOAC might act as a more
effective carrier for the iodine into the leaf. Ethyl iodide and
methyl iodide are not effective as promoters of orange explant
abscission. However, diiodomethane did promote abscission. This may
Indicate the relative ease of release of iodine from the chemical is
a factor.

SUMMARY AND CONCLUSIONS
Investigations were initiated to determine the patterns of
metaboliSfli of labeled lOAC in 'Valencia' and 'Pineapple' sweet orange
leaves, and to test various chemicals for their effects on the rate
of abscission of orange explants. The following observations were made;

1. Both 'Valencia' and 'Pineapple' orange varieties had the same
patterns of labeled metabolites when treated with either IOAC-1-l'^C or
IOAC-2-l'^C. This was the case either with drops applied on the leaf
surface or with petiole uptake.

2. Approximately 3 to 4 times as much foliar-applied lOAC got
into 'Pineapple' orange leaves as compared to 'Valencia' orange leaves.
This difference in uptake was not due to cuticular thicknesses.

3. A major portion of the lOAC (90 to 957o) entering the leaf was
metabolized in both varieties within a relatively short time. The I-C
bond was apparently disrupted and the acetate portion of the molecule
was metabolized to some of the same compounds as was acetate, i.e.
glutamic and aspartic acids. The iodine portion was incorporated into
only 1 compound that was detected. The nature of this compound remains
unknown.

4. NaJ-3lx applied to orange leaves resulted in the same radio-
active compound as did 131 iodine- labeled lOAC .

5. From the testing of chemicals in the orange explant test,
ribose. malonic acid, L-alanine, cysteine, 2 ,4-dini trophenol , potassium
iodide, and diiodome thane accelerated the rate of abscission. Methyl
iodide, ethyl iodide, ascorbic icid , and hexose sugars either retarded
abscission or had no effect.

93

94

It was concluded that the major portion of the lOAC was first
dis rupted at the I-C bond in the orange leaf. This apparently
resulted in free iodine or iodide, and acetate. It appeared that a
large portion of the action of lOAC on orange fruit abscission was
via the iodine and Chat lOAC serves as a carrier for the iodine into
the leaf. Another portion of the action of lOAC could be through the
action of lOAC , per se^, on metabolism since there were products of lOAC
that were not present with acetate and since chemicals known to alter
metabolism, e.g., 2 ,4-dinitrophenol , will hasten abscission. Thus,
there could be an additive effect of lOAC and the iodine arising from
lOAC metabolism.

Also, it appears that the apparent difference in uptake of lOAC
through the surface of the leaves could be the basis for the varied
response between the 'Pineapple' and 'Valencia' orange varieties.

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BIOGRAPHICAL SKETCH

Timothy Joseph Facteau was born in Tupper Lake, New York, on
October 20, 19A0, He received his secondary education at the Peddie
School, Hightstown, New Jersey. He attended Rutgers, the State
University of New Jersey, and received the degree of Bachelor of
Science with a major in Pomology in June, 1963. He was also granted
the degree of Master of Science with a major in Horticulture from
Rutgers, the State University, in June, 1965.

In 1965 he was granted an assistantship from the Department of
Fruit Crops, University of Florida and began study towards the degree
of Doctor of Philosophy which was completed in December, 1967.

He is a member of Alpha Zeta honorary fraternity and a member of
the American Society for Horticultural Science.

He is married to the former Alice Helen Bocchio and they have
two children, Donald Michael and Elaine Michele.

This dissertation was prepared under the direction of the chairman
of the candidate's supervisory committee and has been approved by all
members of that committee. It was submitted to the Dean of the College
of Agriculture and to the Graduate Council, and was approved as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
December, 1967

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Dean, College of Agriculture

Dean, Graduate School

Supervisory Committee;

Co-chairman

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