Metabolism of iodoacetic acid by orange leaves

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Title:
Metabolism of iodoacetic acid by orange leaves
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108 leaves : ill. ; 28 cm.
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Facteau, Timothy Joseph, 1940-
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Oranges   ( lcsh )
Citrus fruits   ( lcsh )
Plant physiology   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1967.
Bibliography:
Includes bibliographical references (leaves 95-106).
Statement of Responsibility:
by Timothy Joseph Facteau.
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Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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notis - ACG2055
oclc - 37767751
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Full Text














METABOLISM OF IODOACETIC ACID
BY ORANGE LEAVES












By
TIMOTHY JOSEPH FACTEAU













A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE LiNIVERSITY OF FLORIDA
[N PARTLAL FLILFILLMENT OF THE REQUIREMENTS FOR THE
DECREE OF DOCTOR OF PHILOSOPHY











UNIVERSITY OF FLORIDA
December, 1967




































Digitized by the Internet Archive
in 2010 with funding from
University of Florida, George A. Smathers Libraries with support from Lyrasis and the Sloan Foundation


http://www.archive.org/details/metabolismofiodo00fact













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.














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












LIST OF TABLES


1. Effect of the time of exposure to IOAC 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 10-4M IOAC-l-l4C for
3 hours ................................................. 38

4. Ethanol extractable radioactivity in extracts of
'Valencia' orange leaf disks treated in 2 ml of
5 x 10-4M IOAC-1-14C for 3 hours......................... 39

5. Radioactivity remaining after ethanol extraction in
'Pineapple' orange leaf disks after treatment in 2 ml
of 5 x 104'M IOAC-1-14C for 3 hours...................... 40

6. Radioactivity remaining after ethanol extraction in
'Valencia' orange leaf disks after treatment in 2 ml
of 5 x 10-4M IOAC-1-14C for 3 hours...................... 41

7. Distribution of radioactivity in fractions from water
extracts of 'Pineapple' orange leaves treated with
IOAC-1-14C............................................... 43

8. Distribution of radioactivity in fractions from water
extracts of 'Valencia' orange leaves treated with
IOAC-1-14C............................................. 44

9. Distribution of radioactivity in fractions of a water
extract of 'Pineapple' orange leaves treated with
IOAC-1-14C ............................................... 45

10. RE values from paper chromatograms of 14C-metabolites
formed by 'Pineapple' and 'Valencia' orange leaves
treated with either IOAC-1-14C or IOAC-2-14C........... 47

11. Distribution of radioactivity in fractions from water
extracts of 'Pineapple' orange leaves treated with
either IOAC-2-14C or acetate-l-14C....................... 49

12. Rf values from paper chromatograms of labeled metabolites
formed by 'Pineapple' orange leaves treated with
IOAC-1-14C.................................. .......... 61






13. Distribution of radioactivity in ethanolic ammonium
and water fractions collected from a Dowex 50-X8
column........... ....................... ................. 64

14. Rf values from paper chromatograms (developed in
butanol solvent) of metabolites formed by 'Pineapple'
and 'Valencia' orange leaves treated with either
IOAC-1-14C or IOAC-2-14C.................................. 70

15. Rf values from paper chromatograms (developed in
methanol solvent) of metabolites formed by
'Pineapple' and 'Valencia' orange leaves treated
with either IOAC-1-14C or IOAC-2-14C..................... 71

16. Rf values from paper chromatograms (developed in
phenol solvent) of metabolites formed by 'Pineapple'
and 'Valencia' orange leaves treated with either
IOAC--114C or IOAC-2-14C................................. 72

17. Rf values from paper chromatograms (developed in
butanol solvent) of metabolites formed by 'Pineapple'
orange leaves treated with either IOAC-1-14C or
acetate-l-14C............................................. 74

18. Rf values from paper chromatograms (developed in
methanol solvent) of metabolites formed by
'Pineapple' orange leaves treated with either
IOAC-l-4C or acetate-l-14C............................... 75

19. Rf values from paper chromatograms (developed in
phenol solvent) of metabolites formed by 'Pineapple'
orange leaves treated with either IOAC-1-14C or
acetate-l-14C.............................................. 76

20. Rf values from paper chromatograms of metabolites
formed by 'Pineapple' orange leaves treated with either
131IOAC or Na131I......................................... 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 IOAC 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. 14CO2 production from attached 'Pineapple' orange
leaves treated with IOAC-2-14C........................... 35

4. 14CO2 production from detached 'Pineapple' orange
leaves treated with IOAC-1-14C or IOAC-2-14C............. 36

5. Autoradiogram of thin-layer separated ether-acidic-
partitioned-fraction of 'Pineapple' orange leaves
treated with IOAC-1-14C................................. 46

6. Autoradiograms of paper chromatographic separation
of 14C-metabolites from 'Pineapple' and 'Valencia'
orange leaves treated with IOAC-1-14C ................... 50

7. Autoradiogram of electrophoretic separation of
14C-metabolites from 'Pineapple' orange leaves
treated with IOAC-1-14C.................................. 51

8. Autoradiogram of 14C-metabolites from 'Pineapple'
and 'Valencia' orange leaves treated with IOAC-1-14C..... 52

9. Electrophoretic separation of 14C-metabolites
resulting from drop application of IOAC-1-14C
to 'Pineapple' orange leaves............................ 53

10. Electrophoretic separation of 14C-metabolites
resulting from drop application of IOAC-1-14C
to 'Valencia' orange leaves.............................. 54

11. Electrophoretic separation of 14C-metabolites
resulting from drop application of IOAC-2-14C
to 'Pineapple' orange leaves............................ 55

12. Electrophoretic pattern of 5 x 10-4M IOAC-1-14C.......... 56

13. Electrophoretic separation of 14C-metabolites
resulting from drop application of IOAC-1-14C
to 'Pineapple' orange leaves............................. 57






14. Autoradiogram of polyamide thin-layer sheet separation
of 14C-metabolites formed by 'Pineapple' orange leaves
treated with IOAC-l-14C................................. 60

15. 14C-metabolites from 'Valencia' orange leaves treated
with IOAC-2-4C ... ............................. .... 65

16. 14C-metabolites from 'Pineapple' orange leaves treated
with IOAC-2-14C........................ ............ 66

17. 14C-metabolites from 'Pineapple' orange leaves treated
with either IOAC-1-14C or acetate-l-14C.................. 68

18. 14C-metabolites from 'Pineapple' orange leaves treated
with IOAC-1-14C......................................... 73

19. 14C-metabolites from 'Pineapple' orange leaves treated
with either IOAC-1-14C or acetate-l-1 C................. 78

20. 14C-metabolites from 'Pineapple' orange leaves treated
with acetate-l-14C...................................... 81

21. 14C-metabolites of a combined sample of IOAC-I-14C and
acetate-l-14C each applied by petiole uptake to 'Pineapple'
orange leaves............................................ 82


vii













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

(IOAC) to whole trees resulted in a loosening of the fruit (76, 77).

The IOAC 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 IOAC if the compound was absorbed

directly through the stem (168). An investigation of absorption of

IOAC 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 IOAC by 'Pineapple' and 'Valencia' orange

leaves in an effort to determine why the 2 varieties varied in their




2

susceptibilities to IOAC. It was also hoped that the study would offer

some clue as to how IOAC acts as a promoter of orange abscission.

Another phase of the study involved the use of an explant test to

screen various chemicals for their effects on rates of abacission.

These chemicals were used because they might offer some ideas as to the

mechanism of abscission.

There are 3 possibilities as to how IOAC could act as an abscission

agent. First, IOAC could act as an enzyme inhibitor since it has been

reported as a sulfhydryl enzyme inhibitor in many systems (88). Second,

some metabolite of IOAC, 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 promoter of abscission (78). This last

possibility would depend on whether or not the I-C bond in IOAC 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 the bean explant test that the maximum rate of

abscission occurs at temperatures between 250 and 300 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 .i 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 many reports on the action of auxin in relation to the abscission

process (1, 3, 4, 13, 15, 16, 102, 136, 155).

One of the early theories on the action of auxin was the "auxin-

gradient" theory (144). Work with beans established the facts that

levels of leaf auxin were higher than levels of stalk 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

applicationseither distal or proximal to the abscission zone were

effective in delaying abscission.

The concentration applied to plants has been shown to influence

abscission. High concentrations of auxin applied to coleus and bean

explants have been shown to inhibit and low concentrations to

accelerate abscission. Whether the applications were proximal or

distal to the abscission zone made no difference.(60). These results

were confirmed (13, 16) and the two-phase theory of the action of auxin

on abscission was proposed.

Further 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 the second

(accelerated by auxin) was called Stage 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 the




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-esterifies 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 (IAA) 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-esterification 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 IAA has resulted in an accelerated rate of methyl

esterification of pectic substances in cell walls of Avena coleoptiles,

but has not resulted in a net change in the final degree of pectic

esterification (89). Also, IAA 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 FME allowed a

polygalacturonase to further break down pectin, thus producing

elasticity and thereby, an increase in cell enlargement (29). Further-

more, IAA 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-esterification), 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

IAA 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

produLtion as suggested (67), then ethylene would probably be a by-

product of pectin breakdown.

Effect of Chemical Treatments on Ethylene Production

The discovery that leaves produced ethylene, that this production

increased as abscission advanced, and that exogenous IAA 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 IAA 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 C02 and

ethylene production, all of which occurred in the mitochondria (117).

Buhler et al. (30) subjected various fruits to ethylene-14C and

found that avocados and pears incorporated 14C from ethylene, but oranges

did not. The amount of 14C 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 320 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 IAA degradation enzymes, peroxidase and IAA 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

IAA. 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 IAA 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 stems 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 14C 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 promoters

of abscission (161). Also, both the D and L forms of leucine, methionine

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).







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

trom 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 these 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 IOAC (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 IOAC-induced abscission of orange

fruits might involve a shift to the pentose shunt (168). IOAC blocks

glycolysis at the triosphosphate dehydrogenase (TPD) step (11, 23, 35,

55, 63, 90) and it would seem likely chat 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 IAA had no effect (82, 83). Others

(142) found that IAA 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, malace dehydrogenase, and

isocitrate dehydrogenase were not affected by the presence of IAA or

kinetin. So, with increasing growth rate (tumor tissue), there was a

decrease in the activities of the pencose shunt. However, it has also

been reported that additions of auxin resulted in an increase in glucose-

6-phosphate dehydrogenase activity (91).

Effects of IOAC 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 et al. (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 the halogen, I Br) Cl (in order of the most to the least effective

inducing abscission) and by specific position occupied (3)2 or 5).

Iodoacetic acid was also found to cause abscission but to a lesser

extent than triiodobenzoic acid.

Effects of IOAC 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 IOAC would inhibit C02 fixation but did not inhibit TPD. In

contrast, it was reported that IOAC inhibited both C02 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 IOAC in the light, the effects of IOAC disappeared.

It was suggested that in darkness, IOAC 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 f keto acid in the pentose shunt. IOAC, therefore,

induced a new pathway of hexose degradation via RUDP (90). IOAC also has

been shown to promote an increase in anthocyanin in 'McIntosh' apples.

It was postulated that IOAC 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 IOAC stimulates glycolysis to a much

greater extent than it stimulates the pentose shunt. Strawberry leaves,

when treated with IOAC, responded by large increases in CO2 production

which was not completely accounted for by losses of sugars and starches.

This increase in C02 production, however, was associated with a rise in

the concentrations of pyruvate and oxaloacetate (9, 10).




19

Further studies of the C6/C1 ratio in strawberry leaves treated

with IOAC indicated that increased glycolysis accounted for the major

part of the stimulation of C02 output. Glucose-6-phosphate (G6P),

fructose-6-phosphate (F6P), and FDP increased greatly. The increases

in G6P and F6P, caused by iodoacetate, were attributed to increased cell

wall permeability such that there was an increase in 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 IOAC acted as an

uncoupler of high-energy phosphate bonds.

lodoacetamide, 2,4-dinitrophenol (DNP), fluoride, arsenite, sodium

bisulfite, and fluoroacetate all were shown to inhibit ethylene production

and respiration in apple tissues (34). The inhibitions caused by DNP

were partially 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 iodoacetamide.

Whether IOAC, as do other abscissing agents including potassium

iodide (137), causes an increase in ethylene production which initiates

abscission; or whether IOAC inhibits ethylene production and, therefore,

abscission accelerated by ethylene, remains to be investigated. Also,

IOAC might act as a SH inhibitor. Michaeles and Schubert (115)

postulated that the reaction R-SH+ICH2COOH--)R S-CH2COOH:-HI was the

mechanism by which IOAC affects SH enzymes. However, Wilson (168) tried

unsuccessfully to histologically determine the effects of IOAC on SH

enzymes in the orange fruit abscission zone.




20

Sweet orange leaves are able to decarboxylate IOAC-1-14C. It was

not ascertained: a) whether the IOAC 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 IAA 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 IOAC 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 IOAC 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 (2-5%) 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 (168, 169). As evidence, it was reported that

IAA 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 oxidation-reduction 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 Cl

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). Protocatechuric, gallic,

cinnamic acid derivatives, coumarins, and others have been shown to be

formed from a shikimic acid pathway (120, 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 et al. (17) were the first to show that fungal cultures could

form an aromatic compound from acetate-l-14C 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-

dimethylphthalide were derived both from methionine and from the methyl

carbon of acetate. Formic acid also contributes C1 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 theB 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 C15 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).






Fatt',' Acid Svnchesis

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 ate also 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, malate

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 14C02 from intact 'Pineapple' orange leaves

treated with IOAC-2-14C was determined by trapping 14002 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

1'C02 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







through the trapping solution.

Closed System

After treatment with either IOAC-1-14C or IOAC-2-14C, 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 Hyamine 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-1-14C, sodium salt, 1.4 mc/m mole

IOAC-2-14C, sodium salt, 6.5 mc/m mole

Acetate-l-14C, sodium salt, 15 mc/m mole

Nal31I, sodium salt, 555.5 mc/m mole

Water was added to make concentrations of 0.1M IOAC and 3 x 10lOM acetic

acid.

13110AC was prepared essentially as described by Conant and Kirner

(45). Nal31I was reacted with 100 mg of monochloroacetic acid in 10 ml

of refluxed, redistilled acetone. The reaction took place at 400 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 -50 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-111-B time-interval

printer, a model C-110-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-100-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, 80% 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 staged with

the results.

Method of Dialysis and Solvent Fractionation

Water extracts were dialyzed using a cellulose acetate membrane

against water at 40 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 fractionational 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 Fractionational Procedures

Water extracts were first freed of most proteins and much of the

pigment material by precipitation with a small amount of 95% ethanol.

After settling, the precipitate was removed by centrifugation at 3980

x g. 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 80% ethanol. Some of the resulting samples were further fractionated

on a Dowex 1-X8 column in the format form.

A set of samples that had been dialyzed was passed through a water-

jacketed 135 x 0.64 cm column of polystyrene S03H resin (type A). The

resin was converted to the ammonium form by 4M ammonium hydroxide. The

column was operated at 660 C with a flow rate of 1.0 ml/min. The UV

absorbance at 290 mu 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 IOAC 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 IOAC in the plant, a

24-hour period was an adequate time interval for the changes to occur.

Therefore, in all experiments where radioactive IOAC 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 14C02 Production from Tissues Treated with IOAC

Both carboxyl and methyl labeled IOAC-14C, when applied to a

limited zone on the surface of 'Pineapple' orange leaves, resulted

in a production of 14CO2. 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).

14CO2 Production from Attached Leaves

The!4CO2 production was periodically sampled at 1-hour intervals

30





31




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



Abscission (')
Chemical Molarity 48 72 96 120 144 168Hrs.


Water control
lodoacetic acid
Mannose

Mannitol

Xylose

Sorbitol

Galactose

Glucono-delta-
lactone
Fructose

Ribose

Dextrose

Ethyl iodide


Methyl iodide


Potassium
iodide

Diiodomethane
Nalonic acid
Cvsteine
Cysteine +
iodoacetic
acid
2,4-dinitrophenol
2,4-dinitrophenol
iodoacetic
acid
L-alanine
L-ascorbic acid


10 50 100


5 x 10-4
5.5 x 10-2
2.8 x 10-2
5.5 x 10-2
2.8 x 10-2
6.6 x 10-2
3.3 x 10-2
5.5 x 10-2
2.8 x 10-2
5.5 x 10-2
2.8 x 10-2
5.5 x 10-2
2.8 x 10-2
5.5 x 10-2
2.8 x 10-2
6.6 x 10-2
3.3 x 10-2
5.5 x 10-2
2.8 x 10-2
10-3
5 x 10-4
10-4
10-3
5 x 10-4
10-4
10-3
5 x 10-4
10-4
10-3
10-3
10-3
10-3
+
5 x 10-4
10-3
10-3
+
5 x 10-4
10-3
5 x 10-4


30
0


20
20
100
70
10
0
100
90
20
30
80
100
0
0
10
20
5
55
30
5
100
70
25
100
80
70


40
20


70
70

100
50
40

100
60
80
80

40
30
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35
5
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85
75

100
100


70
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80
100
100

40
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15
55
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100
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100
90
85
75
75
55
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100
90
80
100
70
40
15


70 100

50 75 100

65 100


15 15 20 30


aOranges harvested


in January, 1967.

































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37

for 24 hours in one test and 48 hours in another test (Fig. 3). The

release of 14C02 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 IOAC is decomposed somewhat by light. However, the level should

have been maintained if that were the case. The peak was obtained

whether or not the plexiglass chamber was darkened.

14C02 Production from Detached Leaves

The 14CO2 production from detached leaves in a closed system

indicated that a small fraction of both IOAC-1-14C and IOAC-2-14C was

metabolized to 14C02 (Fig. 4). Slopes of the 14C02 production curves

show that both the methyl and carboxyl IOAC 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-1-14C showed that in both varieties the amount of

radioactivity extracted with ethanol did not diminish over 48 hours.

This indicated that the IOAC 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 IOAC was not lost as 14C02.

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 IOAC to macro-

molecules or cell wall material.











Table 3. Ethanol extractable radioactivity in 'Pineapple' orange leaf
disks treated in 2 ml of 5 x 10'4M IOAC-l-14C for 3 hours?



Radioactivity in ethanol extract (cpm)b
Hours Exp. I II III IV V

1 11,520 12,350 11,130 8,570 7,480

6 9,730 10,120 8,660 6,110 8,470

10 11,140 12,000 8,260 8,510 9,070

24 12,030 10,520 9,060 9,980 8,040

48 11,300 10,010 9,180 8,700 7,970



aLeaf disks were extracted with 80% ethanol after 1-, 61 10; 24; and 48-
hour intervals following treatment. The specific activity of the
IOAC-1-14C was 1.4 mc/m mole.


bF= .01 N. S. with respect to time of extraction.










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Table 5. Radioactivity remaining after ethanol extraction in
'Pineapple' orange leaf disks after treatment in 2 ml of 5 x 10"4M
IOAC-1-14C for 3 hours.,




Radioactivity in residue (cpm/mg fresh weight)b

Hours Exp. I II 'III IV V

1 2.6 3.0 1.5 1.4 2.0

6 1.8 3.0 1.3 1.9 1.6

10 3.5 2.6 1.9 1.5 1.0

24 2.1 1.3 1.9 0.4 1.8

48 1.7 2.5 0.8 1.6 0.7


aLeaf disks were extracted with 80%
hour intervals following treatment.
IOAC-1-14C was 1.4 mc/m mole.


ethanol after 1, 6, 10-, 24- and 48-
The specific activity of the


bF= 1 N. S. with respect to time of extraction.












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42

Metabolism of IOAC Applied to Leaves in Drops

Both 'Pineapple' and 'Valencia' orange varieties metabolized

labeled IOAC-1-14C. Approximately 60 to 80% of the total radioactivity

absorbed by the leaves could be extracted with water and was dialyzable

(Tables 7 and 8). The average value for the dialyzed components for

the 'Pineapple' variety was 77.9% and that for the 'Valencia' variety

was 63.7%. 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 10% 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 IOAC-

14C 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 IOAC in the Tissues After a 24-Hour Uptake Period

Free IOAC-1-14C in the extracts from orange leaves was removed from

the dialyzed water extract by partitioning between aqueous HC1 (pH 3.0)

and diethyl ether. Only IOAC 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 10% of activity remaining in the leaf) percent of free

IOAC-1-14C 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-1-14C.a




Fraction Total % of
cpm initial value

Basic
Solution Water extract 63,700
pH 8.0 Carbon tetrachloride 0 -
Toluene 0
Benzene 0
Ether 0

Acidic
Solution Etherc 2,460 3.7
pH 3.0 Carbon disulfide 0
Carbon tetrachloride 0
Benzene 0
Toluene 0
Water 67,164 105



aThe IOAC was applied in 2, 10 ul drops, 1 on each side of the main
vein. Time of uptake of IOAC was 24 hours. The water extract was
made basic (pH 8.0) with NaHC03, then adjusted to pH 3.0 with 2N HC1.

bDialyzable portion of extract.


cExtracted with diethyl ether (10 ml, 5 times).










1.0


0.5


0.1

0.0


ETHER ACID FRACTION


IOAC-2-14C


Fig. 5. Autoradiogram of thin-layer separated ether-acidic-
partitioned-fraction of 'Pineapple' orange leaves treated
with IOAC-1-14C. The IOAC was applied in 2, 10 y 1 drops,
1 on each side of the main vein. Developed in butanol:
ethanol:water, 65:10:10, v/v/v, on Eastman chromagram
silica gel sheets (type K 301R2).


6::::


IOAC-1-14C


00





47






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48

'Pineapple' leaves treated with IOAC-2-14C also had approximately the

same percentage of free IOAC in the ether fraction (Table 11). These

results indicated that most of the IOAC 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 IOAC resulted in the same 14C distribution

pattern, it appeared that the 2 carbons were metabolized similarly.

Carboxyl and Methyl 14C-labeled IOAC Metabolites

When carboxyl or methyl 14C-labeled IOAC was applied to 'Pineapple'

and 'Valencia' orange leaves, both metabolized approximately 90% of the

absorbed IOAC 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-1-14C or IOAC-2-14C

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-1-14C and IOAC-2-14C

(Figs. 8-11). IOAC 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-1-14C or IOAC-2-14C. 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




















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'PINEAPPLE' IOAC-1-14C 'VALENCIA'

Fig. 6. Autoradiograms of paper chromatographic separation
of 14C-metabolites from 'Pineapple' and 'Valencia' orange
leaves treated with IOAC-1-14C. The IOAC was applied in
2, 10 yl drops, 1 on each side of the main vein. Time
of uptake of IOAC was 24 hours. Chromatogramed on Whatman
#3 paper and developed in N-propanol:water, 6:4, v/v. The
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1 P. 14O4
J-'d 'JO



O C.44-4O0.
00 4 .9









i8n
rZ4U
440C C4I
wI r










ANODE








ORIGIN



























CATHODE


'VALENCIA' 'PINEAPPLE' 'PINEAPPLE' 'PINEAPPLE'

Fig. 8. Autoradiogram of 14C-metabolites from 'Pineapple'
and 'Valencia' orange leaves treated with IOAC-1-14C. The
IOAC was applied in 2, 10 1l drops, 1 on each side of the
main vein. Time of uptake of IOAC was 24 hours. Separated
by paper electrophoresis in 0.4N acetic acid buffer, pH 2.5
for 4 hours at 500 volts.



















500 %
\

1'






300


C- I






100
I










CATHODE ORIGIN ANODE

Fig. 9. Electrophoretic separation of 14C-metabolites resulting
from drop application of IOAC-1-14C to 'Pineapple' orange leaves.
Developed for 4 hours at 500 volts in 0.4N acetic acid buffer,
pH 2.5.
pH 2.5.


















300 \


1\



210 \

I
I



120
I




yII I


II /
30


CATHODE ORIGIN ANODE

Fig. 10. Electrophoretic separation of 14C-metabolites resulting
from drop application of IOAC-1-14C to 'Valencia' orange leaves.
Developed for 4 hours at 500 volts in 0.4N acetic acid buffer,
pH 2.5.


















300 %






210






120






300
CATHODE ORIGIN ANNODE
I / '

I
II I /
30 /



CATHODE ORIGIN ANNODE

Fig. 11. Electrophoretic separation of 14C-metabolites resulting
from drop application of IOAC-2-14C to 'Pineapple' orange leaves.
Developed for 4 hours at 500 volts in 0.4N acetic acid buffer,
pH 2.5.





















300







210







120 o 1








30
I









CATHODE ORIGIN ANODE

Fig. 12. Electrophoretic pattern of 5 x 10-4M IOAC-1-14C.
Developed for 4 hours at 500 volts in 0.4N acetic acid buffer,
pH 2.5.


















300



II'

4'

210
I



I
I
120 I
I
I
I
I


30 III


CATHODE ORIGIN ANODE

Fig. 13. Electrophoretic separation of 14C-metabolites resulting
from drop application of IOAC-1-14C 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 IOAC.

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 IOAC. 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 IOAC-1-14C

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, further

separations by thin-layer chromatography of these radioactive fractions

indicated that 14C labeled sugars, flavonoid or phenolic compounds (Fig.

14) were not formed as a result of the metabolism of IOAC. 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 IOAC 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. Autoradiogram of polyamide thin-layer sheet separation
of 14C-metabolites formed by 'Pineapple' orange leaves treated
with IOAC-1-14C. 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


8- 130


9- 131-134

10- 135

11- 140

12- 141-142


13- 143-145

14- 146-147

15- 148-149

16- 181-182

17- Naringin






60












%-







-I
r-4








r-4



















a%





VA
-4















-4







04
I-4

























c c
C-4










Table 12. Rf values from paper chromatograms of labeled metabolites
formed by 'Pineapple' orange leaves treated with IOAC-1-14C?


Rf values of labeled metabolites
Solvent systems

1 2 3

Fraction 115 .1, .20 .12, .32 .17, .31

Fraction 121-124 .17 .10, .31 .05, .12, .31

Aspartic acid .16-.22 .14 .17

Glutamic acid .22-.28 .27 .33


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

bl Butanol:water: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.




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-1-14C. 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 IOAC 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-l-14C in the same

manner as those treated with IOAC-14C 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 IOAC 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-l-14C.




63

Metabolism of IOAC Applied to the Leaves by Petiole Uptake

Amount of Free IOAC Remaining in the Tissues

The amount of unmetabolized IOAC remaining in the 'Pineapple' and

'Valencia' leaves after uptake for 2 hours was found to be not greater

than 30% of the total extractable radioactivity. This was calculated

from the total radioactivity in the acidic and neutral fractions(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 IOAC (Rf .72-.74) represented a small fraction

of the 30% radioactivity in the acidic and neutral fraction. This

compared favorably with the approximate 5% free IOAC 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 formatt) with subsequent thin-layer

separation on Eastman chromagram silica gel films, showed that labeled

compounds appeared in the water washes and in the 1% 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 IOAC

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

IOAC-1-14C or IOAC-2-14C 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 column.



Percent radioactivity in fractions
Exp. I II
Sample Water Ammonium Water Ammonium

'Pineapple' acetate-l-14C 86 14 80 20

'Valencia' acetate-l-14C 83 17 89 11

'Pineapple' IOAC-I-14C 59 41 23 77

'Valencia' IOAC-1-14C 16 84 29 71

'Pineapple' IOAC-2-14C 21 79 33 67

'Valencia' IOAC-2-14C 33 67 16 84

'Pineapple' 131IOAC 98 2 98 2



aExtracts of 'Pineapple' and 'Valencia' orange leaves in which either
IOAC-1-14C, IOAC-2-14C, 131IOAC, or acetate-l-14C were applied by
petiole uptake. Uptake time was 1 hour, after which the leaves were
placed in water for an additional hour.







65




















c'J 1
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66




















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Fig. 17. 14C-metabolites from 'Pineapple' orange leaves
treated with either TOAC-1-14C or acetate-l-14C. 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 formatt) 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-1-14C,

2 acetate-l-14C,

3 IOAC-1-14C,

4 acetate-l-14C,

5 IOAC-1-14C,

6 acetate-l-14C,

7 IOAC-1-14C,

8 acetate-l-14C,


water

water

water

water

water

water

water

water


fraction,

fraction,

fraction,

fraction,

fraction,

fraction,

fraction,

fraction,


2, 3, 4, water washes.

2, 3, 4, water washes.

1% formic acid elution.

1% formic acid elution.

5% formic acid elution.

5% formic acid elution.

40% formic acid elution.

40% formic acid elution.


















































4 5 6 7 8


0.9


0.5


0.0


1 2 3




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 ninhydrin. 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 format column. These

fractions from the format column were further separated by paper

chromatography using 3 solvent systems. Again, in the ammonium fraction

sample.from the Dowex 50-X8 column fractionatedd on the format 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 format 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 formatt) 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 1% formic acid elution (Fig.

19, number 3). These components also gave the correct ninhydrin colored


















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a) Ir< 0~ I n- 01 n0 O C 0
0l p GL U 4.1 4Y- c) 4J flC! C! u c S' S.4

.0 P n r 0 o 0. -4 t CU
CU CU 0 0 02 u4~4E-






Fig. 19. 14C-metabolites from 'Pineapple' orange leaves treated
with either IOAC-1-14C or acetate-1-14C. 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 formatt) 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-1-14C,

2 acetate-l-14C,

3 IOAC-1-14C,

4 acetate-l-14C,

5 IOAC-1-14C,

6 IOAC-1-14C,

7 glutamic acid.

8 aspartic acid.


1st water wash.

1st water wash.

ammonium fraction,

ammonium fraction,

ammonium fraction,

ammonium fraction,


1% formic acid elution.

1% formic acid elution.

5% formic acid elution.

2, 3, 4 water washes.


9 alanine.

















































2 3 4


5 6 7 8 9


0.9r


0.5


0.0


1




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 positive(Fig. 19, number 2) compound obtained from the

metabolism of acetate by 'Pineapple' leaves. Two metabolites also

were present in the 5% 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 Rf 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-l-14C 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 75% of 14C-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 80% 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-14C 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 format

column. Two of these metabolites (in the ammonium fraction) were ninhydrin





80

positive and corresponded to glutamic and aspartic acids, as did the

IOAC-14C metabolites.

Thin-layer chromatography of the ammonium fraction (from the Dowez

50-X8 column) further eluted from a format column, showed that 2

ninhydrin-positive, radioactive metabolites were present in the 1%

formic acid eluding fraction (Fig. 19, number 4) with similar Rf values

to those of glutamic and aspartic acids. The first water wash from the

format 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 format column showed that at least 1

metabolite was present in the 5% 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 40% 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 IOAC

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 IOAC produced some compounds similar to those formed when acetate was

metabolized, it would appear that some of the IOAC was metabolized as

acetate. However, it was clear that there was a difference between the

metabolism of IOAC and that of acetate in 'Pineapple' leaves.






81



























C %0 4J -1 r-4
*r4 00u

,a 0 0
4.) rd
0 wu d >1
( 04 O -0





ni> wa)


44 e
00 4 0> t
X -4 -
o' 0) *4J 0 W





I i0 4-i
d :-i 0)l x
-4 (U
0 -4 -4
4 p. 1-1 ( 4-1 in
S 0o e




e u a 0
0 w 0 C0
4 w -

H E a)
S E-4l 0 u 0




-4 W4


u w 41 -4
S4-J 0 (4
*, C U -'

.0 E-l4 0. u
cd B *-4 M
J 0 C
W U 0 10







--44
S E *






U* CQ. *r4 XC
-------- 00 0 :3 &3: 0
*r4
____________ br


wao





82
























0 co


Cd OO4 3 4
SU U


o 01
mP2




r 0 0-4 -
I a) r4
ao ed




0 U (fl



0 CO


H -4 0
o4 p





.,.
0 z u


0 0 r-0
o z0

O 0 -
\ U O( -



M 4*-1 0 C










0 0 C
'-. m- 0









m 0 >3
H 0 0
4-4 Q-, i-. 0o

I 0 '> .u




M a
-4 -4 -J






Mj 0
D IUo c
u u$l







CV 0
_______ n- 4






Metabolism of 131IOAC

131IOAC treatment of 'Pineapple' leaves resulted in a compound

with a different Rf value that any formed in the IOAC-14C 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 Nal311

Sodium 1311 treated 'Pineapple' leaves resulted in a metabolite

with an Rf value similar to the 131I0AC compound (Table 20). Approximately

30% 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).











Table 20. Rf values from paper chromatograms of metabolites formed
by 'Pineapple' orange leaves treated with either 13110AC or
Na131 a





Rf values of labeled metabolites

Solvent systems

Sample 1 2 3

13110AC .85 .70-.72 .64

'Pineapple' 13110AC water .34-.38 .60 .26
fraction

'Pineapple' Nal31 water .32-.34 .58 .23
fraction



aThe 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.


bI-
2 -
3-


Butanol:water:acetic acid, 4:5:1, v/v/v (upper phase).
Methanol:water:acetic acid, 19:1:1, v/v/v.
Phenol:water, 100:20, v/v, in 0.04% 8-hydroxyquinoline.












Table 21. Distribution of radioactivity in fractions from 'Pineapple'
orange leaves treated with sodium 131iodide and eluted from a Dowex
50-X8 columnO


Fraction % of total radioactivity


Ethanol precipitated 34

Water 63

Ammonium 3



aThe 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

IOAC. In both instances, the patterns of labeled metabolites formed

from IOAC-14C were the same. This was the case whether the IOAC was taken

into the leaf through the surface or through the petiole. There was a

greater quantity of IOAC-14C 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 IOAC in the petiole uptake tests

was different in some respects simply because of the route of entry of

the IOAC.

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 IOAC after only 2 hours. Also, the same results,

namely, only a small percentage of free IOAC, were found with the surface

application experiments. The duration of this test was 24 hours.

There was no significant incorporation of 14C 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 0.1M IOAC. Absorption rates of IOAC appeared to be

slow under these conditions and not as much IOAC 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 1% formic acid elution of a

Dowex 1-X8 column formatte. 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 formatt) 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 14C labeled IOAC treated orange leaves produced

14CO2 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 IOAC 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 IOAC. Again,

labeled glutamic and aspartic acids were present in both cases. Also,

labeled alanine was tentatively identified as a component arising from

acetate-l-14C metabolism.

Other labeled compounds appeared with IOAC that were not present

with acetate. A portion of the IOAC could have been metabolized

differently, or could have affected metabolism, per se. However, it

was clear from the C02 pattern and the labeled metabolite patterns of

acetate and IOAC that a large portion of the IOAC was broken at the

I-C bond.

The metabolic patterns of 131IOAC and IOAC-14C indicated further

that this disruption occurred in at least 90 to 95% of the IOAC in the

tissue. The time of bond separation was not readily apparent. However,

light was not the agent that catalyzed this reaction. Tests of IOAC-14C

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. IOAC does decompose in light but the rate is

apparently very slow. It has been calculated to be 0.05%/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 13110AC or

sodium 131iodide. The identity of this compound was unknown but it

was not free 131iodine. The component was not retained on a Dowex




89

50-X8 column. About 30% of the radioactivity with 13110AC treated

leaves was precipitated by alcohol. This could indicate that the

131iodine was bound to a large molecule, probably protein. Since

potassium iodide will promote orange explant abscission, it appears

that the iodide portion of IOAC could be responsible for a portion of

the abscission-accelerating activity. The IOAC may act as a better

carrier for iodine since potassium iodide in a spray will not promote

orange abscission but IOAC 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 IOAC 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 IOAC. 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 IOAC by 'Pineapple' leaves or a slower rate of translocation. A

previous investigation on IOAC 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 IOAC 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 14C from IOAC 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 IOAC on orange fruit abscission. The IOAC was metabolized

very quickly in the tissues; therefore, it would seem that the action

on abscission, per se,was not entirely through the action of intact

IOAC 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 IOAC acts as an uncoupler

of high-energy phosphate systems (47). Dinitrophenol acts similarly

(46); therefore, the effect of some of the IOAC might be on high-energy

transfer systems. Also,there could be an additive effect of the iodine

and the unmetabolized IOAC.

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 10'3M.

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 IOAC

only a slight reduction in the abscission rate produced by IOAC alone

was found. If IOAC acted as a sulfhydryl enzyme inhibitor, possibly

flooding the tissues with sulfhydryl material might have reduced the

effect of IOAC. There was no interaction between cysteine and IOAC 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 IOAC probably are not the active

agents in initiating the events leading to abscission. Since some

products from metabolism of IOAC 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 IOAC 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.