. .
a

 

THE EFFECT OF TEMPERATURE ON
ETHYLENE EVOLUTION FROM ETHEPHON
TREATED SOUR CHERRY LEAVES
(PRUNUS CERASUS L., CV MONTMORENCY)
AND FROM BUFFERED ETHEPHON
SOLUTIONS

Thesis for the Degree of M. S.
MICHIGAN STATE U'WERSITY
WILLIAM CHARLES OLIEN
1976

I III; WIT III II 111 III III [III “I u I,

LIBRAR 1!
Stage

 

 

 

 

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I

ABSTRACT

THE EFFECT OF TEMPERATURE 0N ETHYLENE EVOLUTION
FROM ETHEPHON TREATED SOUR CHERRY LEAVES
(PRUNUS CERASUS L., CV MONTMORENCY) AND
FROM BUFFERED ETHEPHON SOLUTIONS

 

by

William Charles Olien

The effect of temperature on ethylene evolution from ethephon

treated sour cherry (Prunus cerasus L., cv Montmorency) leaves and from

 

buffered ethephon solutions was evaluated. Uniform, fully expanded
leaves were treated in the field with l25 ug of ethephon, 2-(chlor-
ethyl)phosphonic acid, applied in 5 drops (5 ul each) to the upper
leaf surface. After 24 hours, leaves were detached and ethylene evo-
lution was measured after incubation at temperatures between 10 and
34°C. Ethylene evolution was monitored from ethephon solutions of the
same dose (125 ug) buffered between pH 2.6 and 7.0 with citrate-
dibasic sodium phosphate buffer. The rate of ethylene evolution from
treated leaves increased markedly with temperature, with an "Ea" of

about 30 Kcal mole-1

(Q10:lO—20°C of about 7). A comparable tempera-
ture effect was observed for buffered ethephon solutions, regardless
of pH. Endogenous ethylene is generally evolved over 0 to 30°C with

l

William Charles Olien

an "Ea“ in the range of 14 Kcal mole-1 (Qlole-20°C of about 2). The
rate of ethylene evolution from ethephon treated leaves was found to
be completely and quickly reversible with a reversal in temperature.
The rate of ethylene evolution from buffered ethephon solutions at
20°C increased exponentially with an increase in pH and was not
associated with the pKa values apparent from the titration curve

for ethephon.

THE EFFECT OF TEMPERATURE 0N ETHYLENE EVOLUTION
FROM ETHEPHON TREATED SOUR CHERRY LEAVES
(PRUNUS CERASUS L., CV MONTMORENCY) AND
FROM BUFFERED ETHEPHON SOLUTIONS

 

by

William Charles Olien

A THESIS

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

MASTER OF SCIENCE
Department of Horticulture

1976

ACKNOWLEDGMENTS

I wish to thank Dr. M. J. Bukovac for his assistance,
guidance, and support during the course of my graduate program.
Sincere appreciation is extended to Dr. F. G. Dennis, Jr. and
Dr. L. F. Wolterink for their helpful suggestions and for serving

on my examining committee.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES ......................... iv
LIST OF FIGURES ........................ vi
LIST OF APPENDICES ....................... vii
INTRODUCTION .......................... l
METHODS AND MATERIALS ..................... 19
RESULTS ............................ 28
DISCUSSION ........................... 47
CONCLUSIONS .......................... 54
APPENDICES .......................... 56
LIST OF REFERENCES ....................... 66

LIST OF TABLES

Table Page

l. Effect of temperature on endogenous ethylene evolution. . 13

2. Effect of temperature on rate of ethylene evolution from
control and ethephon treated tomatortissue ....... 15

3. Effect of temperature on rate of ethylene evolution from
ethephon treated leaves, first experiment ....... 29

4. Effect of temperature on rate of ethylene evolution from
ethephon treated leaves, second experiment ....... 30

5. Rate of ethylene evolution at 26°C from ethephon treated
leaves as affected by preliminary temperature
treatment . . . . . . . . . . . . . . . . . . . . . . . 33

6. Effect of constant vs alternating temperatures on rate
of ethylene evolution from ethephon treated sour
cherry leaves ..................... 35

7. Effect of temperature and time on ethylene evolution
from ethephon treated leaves .............. 36

8. Effect of temperature on rate of ethylene evolution
from 'Napoleon' sweet cherry shoot tissue collected
before bud swell .................... 38

9. Effect of temperature on rate of ethylene evolution
from ethephon solutions buffered at pH 6.0 ....... 39

l0. Effect of temperature and pH on the rate of ethylene
evolution from buffered ethephon solutions ....... 4]

iv

LIST OF TABLES (c0nt'd.)

Table Page

ll. Effect of ethephon concentration on rate of
ethylene evolution ................... 42

LIST OF FIGURES

Figure Page
1. The relationship of 010 and Ea .............. ll
2. Temperature effect on rate of ethylene evolution from

buffered ethephon solutions, control tissues, and

ethephon treated tissues ................ 32

3. The effect of pH on rate of ethylene evolution from
buffered solutions of ethephon held at 20°C ...... 44

4. Titration curve for l00 ml 0.0lN ethephon treated with
0.lN NaOH ....................... 46

5. Estimated limits on the Arrhenius slope for rate of
ethylene evolution from ethephon treated tissue . . . . 50

vi

LIST OF APPENDICES

Appendix Page
A. THE ARRHENIUS EQUATION ................. 56

B. DETERMINATION ()F ETHEPHON DOSE PER LEAF FOR ADEQUATE
ETHYLENE EVOLUTION .................. 58
C. ETHEPHON LEAF PHYTOTOXICITY ............... 60

D. DETERMINATION OF THE AMOUNT OF ETHYLENE SOLUBLE IN THE
WATER PRESENT DURING ETHYLENE ACCUMULATION ...... 61

E. RELATIVE SENSITIVITY 0F QUIESCENT SHOOT TISSUE T0
ETHEPHON AMONG SEVERAL TREE FRUIT SPECIES ....... 63

vii

INTRODUCTION

Cherry Harvest

 

The cherry industry is important to the economy of Michigan.
Michigan is first in the nation in sour cherry production and fourth
in sweet cherry production (Michigan Agricultural Statistics, l975).
In l974, the total cherry crop was valued at roughly 45 million
dollars.

Harvest is one of the most expensive operations in cherry
production (Childers, l973). Because of this, shake-and-catch machine
harvest was developed in the early l960's, and the practice was
rapidly adopted by the growers (O'Brien, l969). This resulted in a
tremendous increase in efficiency and economy of the sour cherry har-
vest operation (Childers, l973). The shaking force required to har-
vest mature sweet cherries was too great, however, and resulted in a
high percentage of bruised fruit and severe damage to the tree (Whit-
tenberger and LaBelle, l969). For this reason, mechanical harvest was
practical only for sour cherry. Damage to sour cherry trees was also
apparent. The average life of a sour cherry tree has declined from

30-40 years to 25 years since the advent of shake-and-catch harvest

(Childers, l973). In addition to tree damage, other limitations are
associated with the use of mechanical harvesters. The population of
fruit on a tree does not mature uniformly, and machine harvest is not
selective for mature fruit. The grower has limited control over fruit
maturity and so has a limited capacity to program harvest.

More efficient transfer of the shaking force through the
tree, and less bruising of fruit were attained with pruning practices
more appropriate to machine harvest (Childers, I973). The problems
of programing harvest, variation in fruit maturity, and the high fruit
removal force requiring a shaking energy damaging to the tree still re-
mained (Bukovac, et al., l97lb; Wittenbach and Bukovac, l972b). Be-
cause of strong varietal preferences of the market, and the long
period of time required to develop new cultivars, plant breeding does
not offer a solution to these problems at this time (Probsting, l973).

The use of plant hormones has provided an alternative solution.

CherrygFruit Abscission

Removal of mature fruit from the tree is dependent on the
development of an abscission layer between the fruit and the pedicel
(Bukovac, l97l). Ethylene plays a critical role in the abscission of

both sweet and sour cherry (Wittenbach and Bukovac, T974).

A two-phase abscission process is visualized for sour cherry,
similar to that proposed for leaf abscission in bean (Wittenbach and
Bukovac, l973). The development of the sour cherry abscission layer
cannot be induced by ethylene much before stage 111 (final swell) of
fruit growth. Induction of the layer probably occurs between Stages
II (pit hardening) and III. Ethylene may shorten the duration of
the induction phase, possibly affecting IAA levels by stimulation of
peroxidase activity, but must be present in the developmental phase
for abscission to occur (Wittenbach and Bukovac, I974). The effect
of ethylene is thought to be direct in the abscission layer develop-
ment. Ethylene appears to promote the activity of hydrolytic en—
zymes in the cell wall (Bukovac, et al., l97lb), and has been asso—
ciated with both the synthesis and secretion of cellulase (Abeles,
l973; Wittenbach and Bukovac, l973).

Abscission in sweet cherry is also accelerated by ethylene in
stage III of fruit development (Wittenbach and Bukovac, l973). The
effect seems to be associated more with fruit ripening, however, than
with abscission layer formation (Wittenbach and Bukovac, l972b, l973).
During pericarp expansion and fruit ripening mechanical forces result
in cell wall shearing and separation (Bukovac, et al., l97lb; Witten-
bach and Bukovac, l972b), forming an abscission layer as defined by

Esau (I965) and modified by Wittenbach and Bukovac (l972a). Cell wall

changes in pectins, non-cellulosic polysaccharides, and cellulose were

not noted in sweet cherry (Wittenbach and Bukovac, I972a, l972b, l973).

Ethephon in Cherry Fruit Abscission

 

Because of the important role ethylene plays in the abscis-
sion process, means were sought to deliver a controlled dose of ethy-
lene to the abscission zone at the time desired. Direct application
of ethylene gas under orchard conditions would be impractical. Many
chemicals such as auxin, ABA, cycloheximide, ascorbic acid, and
ethylene itself, induce endogenous ethylene evolution (Cooper, et al.,
I968; Bukovac, et al., I969). Other compounds, such as betahydroxy-
ethylhydrazine and ethephon, 2-(chloroethyl)phosphonic acid, release
ethylene on degradation (Warner and Leopold, I967; Morgan, I972). Of
these compounds, ethephon has been most useful (Bukovac, l97l; Morgan,
l972; Abeles, I973). The chemical structure of this compound is as

follows:
0

CICHZ-CHz-P-OH
OH

The mechanism of ethephon degradation originally proposed by Maynard
and Swan (I963) and later supported by Yang (I969) involves a nucleo-

philic attack of water or hydroxide ion on the phosphorus moiety with

the concerted elimination of chloride, releasing ethephon as a product.
Ethephon is probably nonenzymatically degraded in plant tissue

(Dennis, et al., I970). Rate of ethephon degradation and the corre-
sponding release of ethylene from aqueous solution is strongly affected
by pH (Edgerton and Blanpied, I968; Anonymous, I969; Bukovac, et al.,
I969; Warner and Leopold, l969). Ethephon appears stable below pH 3.0
or 3.5 and degrades at an increasingly rapid rate with an increase in
pH.

Initial testing of ethephon for promotion of cherry fruit
abscission indicated that fruit removal force was decreased and that
fruit maturity was advanced and more uniform within three days after
application of a 500 to 4000 ppm dilute spray (Bukovac, et al., I969).
Maximum effect was noted after six days. In addition, a delay in
anthesis the following spring was observed with the higher concentra-
tions (2000 and 4000 ppm).

Ethephon was cleared for commercial cherry production in
I973 and has made mechanical harvest of sweet cherries practical for
the first time. Ethephon must be applied in stage III of fruit growth
because of the nature of the fruit ripening and abscission processes.
Current recommendations for use may be found in the I976 Fruit Pesti-
cide Handbook and on the current product label. Generally, a 200 ppm

dilute spray applied 7 to ID days before the projected havest date is

recommended for sour cherry. A dilute application of from 250 to 400
ppm applied 7 to l4 days before harvest effectively loosens sweet
cherries.

The development of ethephon as a fruit abscission compound
was an important advance in the mechanical harvest of cherries. How-
ever, phytotoxicity problems have been noted (Bukovac, et al., I969)
at higher rates (2000 and 4000 ppm) on sweet and sour cherry and plum.
These included leaf yellowing, leaf abscission, terminal dieback,
gummosis, and enlarged lenticels on current season's wood. In gen-
eral, these effects were most severe on trees of low vigor and on weak
wood in the central part of the tree. Similar effects have been re-
ported by Anderson (I969) andhfilde and Edgerton (I975) for sour cherry,
by Bradley, et al. (I969) for apricot, and by Buchanan and Biggs
(I969), Proebsting and Mills (I969), Rom and Scott (I97l), Stembridge
and Gambrell (I97l), and Daniell and Wilkinson (I972) for peach.

Phytotoxicity varies among species (Bukovac, et al., l969).
We compared the sensitivity of five tree fruit species in a study of
bud expansion, gummosis, internal browning, and ethylene evolution by
holding the bases of one-year-old shoots, collected April, I975
shortly before bud swell, in 0, 50, ICC, and 200 ppm ethephon solu-
tions (Appendix 5). Species could be ranked from least to most sensi-

tive as follows: apple (Malus domestica Bork., cv. McIntosh), sour

 

cherry (Prunus cerasus L., cv. Montmorency), sweet cherry (Prunus

 

avium L., cv. Windsor), plum (Prunus domestica L., cv. Stanley). and

 

peach (Prunus persica L., cv. Redhaven).

 

Phytotoxicity also varies from year to year (Wilde and
Edgerton, 1975). Field observation has indicated that temperature
is an important factor in the performance of ethephon (1976 Fruit
Pesticide Handbook). In the summer of 1973, growers in Michigan
experienced severe gummosis on ethephon treated cherry trees, and
part of this was attributed to temperatures of 90 to 95°F for several
days after ethephon application (Bukovac, personal communication).
Gummosis in tulip bulbs is affected by temperature before and during
exposure to ethylene (Kamerbeck, et al., 1971). The recommended temp-
erature range for ethephon application is 15.5°C (60°F) to 29.4°C
(85°F). The effect of temperature is, of course, continuous through
this range. Temperature appears to be important not only during the
absorption period, but also for several days thereafter, as most of
the compound degrades. While earlier application may be substituted
for higher concentration when using ethephon as an aid to cherry har-
vest (1976 Fruit Pesticide Handbook), temperature must also be con-

sidered in determining the dose for a desired response.

Temperature Effect on Rate of
Ethylene Evolution

The dependence of reaction rate on temperature is often
studied in terms of the Arrhenius equation. The derivation and limi-

tations of this equation are presented in Appendix 1. Briefly, the

equation describes a line where I, the integration constant, and %53

the slope, are constant parameters over the temperature range investi-

gated.

Equation 1 1n k = I - %5 T"1 k rate constant for the forward

or reverse reaction

E = activation energy for the
forward or reverse reaction

R = ideal gas constant

T = absolute temperature

The dependence of reaction rate on temperature over a linear
portion of such a plot is expressed by Ea’ which is directly propor-
tional to the slope. The energy of activation for a single reaction
is the energy limit below which reactants can not be converted to
products. Individual reactant molecules are distributed over a range
of energy values. As heat energy is put into the system, the tempera-
ture (mean translational kinetic energy) increases and the portion of

reactant molecules with energy above the activation limit increases.

This plot (Equation I) may be used for homogenous gas reactions,
reactions in solution, and heterogeneous PFOCGSSES- Thus, the slope
and Ea may actually reflect several simultaneous reactions. To empha-
size this, I will refer to the activation energy derived from the
regression of an Arrhenius plot as "Ea".

Another method of expressing the effect of temperature on
reaction rate is 010. 010 is defined as the ratio of the reaction
rate at T + 10°C over the reaction rate at T°C. This ratio and var-
ious equations used to estimate this ratio are based on T and not
T']. For this reason, even for a linear Arrhenius plot, the value
of 010 depends on the temperature interval selected. For an arbitrary
linear Arrhenius plot (Figure I), Ea is constant regardless of temper-
ature, but 010 is constant only for a given temperature interval.
Since 010 is attempting to indicate the 510pe of this plot, the pre-
dicted rates from the regression equation (Equation I) are better
estimates to use in calculating 010 than the rates obtained experi—
mentally. Since 010 is often used in the literature, I will also
give the 010 value derived for a given temperature interval from the
regression equation of the Arrhenius plot as Q103T1‘T2°C- Experi-

ments can then be compared by reference to either the defined 010

or E .
a

I0

FIG. l.--The relationship of 010 and Ea'
Note that 010 varies with the temperature interval selected

for an arbitrary linear Arrhenius plot.

II

  
  
  
  
 

4.6 ~ 0'0140-50°C=5.I7

0.0: 30-40% = 5.92

 

 

0
«0- 2.3-
O
a:
I:
0.0: 20-30°C= 7.00
oI
0,01 l0-20°C =7.78
Ea: 33.72 Kcal/mole
_2.3 l n 1 1 n T
3.096 3.l95 3.300 3.4!3 3.534 (°K"xl0)
50 40 30 20 10 (‘CI
Temperature

Figure I

12

Few investigators have examined the effect of temperature
on the rate of endogenous ethylene evolution in higher plants. Only
two publications imply an analysis of the data in mentioning 010
(Hansen, 1945; Burg and Thimann, 1959), and a value for 010 is given
only by Burg and Thimann (1959). Data for the effect of temperature
on endogenous ethylene production, or approximations where necessary
were obtained from five papers (Hansen, 1942, 1945; Biale, et al.,
1954; Burg and Thimann, 1959; Lougheed and Franklin, 1972) and two
unpublished sources (0. C. Coston; M. SaItveit). The experiments
involved both fruit (pear, apple, cherimoya, and tomato) and vegeta-
tive tissue (subapical etiolatedpea sections and tomato foliage).
Values calculated for 010:10-20°C varied from 1.49 to 4.37 with a
mean of 2.53 t 0.78; values for "Ea" ranged from 6.5 to 17.4 with a
mean of 14.64 t 4.83 Kcal mole'1 (Table I). This information can
not be taken as a critical evaluation of the effect of temperature
on endogenous ethylene evolution, but it does indicate that the
general trend is in the expected 010 range of 2.

Maximum rates of ethylene production are obtained between
20 and 30°C (Hansen, 1942; Burg and Thimann, 1959; Lougheed and Frank-
lin, 1972). Ethylene evolution was inhibited above this temperature
and was completely inhibited at 40°C (Hansen, 1942; Burg and Thimann,

1959). This inhibition is, to some extent, reversible (Burg and

Table 1. Effect of temperature on endogenous ethylene evolution.

13

I

 

 

 

 

 

Q II E II

10 a

Plant, Tissue (IO-20°C) (Kcal mole-1) Reference

Pear, fruit 1.95 11.0 Hansen, 1942

Cherimoya, fruit 1.49 6.5 Biale, et al., 1954

Apple, fruit 2.86 17.4 Hansen, 1945

Apple, fruit 4.37 24.4 Hansen, 1945

Apple, fruit 2.82 17.1 Hansen, 1945

Apple, fruit 2.24 13.4 Burg & Thimann, 1959

Apple, fruit 2.65 16.0 Coston, 1976
(unpublished)

Tomato, fruit 2.75 16.6 Lougheed & Franklin,
1972

Tomato, foliage 2.08 12.0 Lougheed & Franklin,
1972

Peas, subapical

section 2.09 11.8 Saltveit, 1976
(unpublished)
mean 2.53 i 0.78 14.6 T 4.8

 

1Calculations were made from the reported rates of endogenous
ethylene evolution at various temperatures.

I4

Thimann, 1959; Burg, 1962). The lower temperature limit for endogen-
ous ethylene evolution has not been investigated, but Hansen (1942,
1945) showed ethylene evolution in pear and apple at 0°C. Ethylene
evolution from Bosc pear was enhanced at room temperature by storage
for the preceding week at 5 or 10°C (Sfakiotakis and Dilley, 1971).
The effect was not as pronounced when pretreated at 0°C. Freeze in-
duced ethylene was detected in tender orange leaves subsequently
monitored at room temperature (Young and Meredith, 1971).

Little work has been done on the effect of temperature on the
rate of ethephon degradation. Lougheed and Franklin (1972) studied
this relationship using ethephon treated tomato fruit and foliage
under both air and N2 atmospheres. Calculated values based on their
data are summarized in Table 2. Ethylene was measured 24 (fruit) or
4 (foliage) hours after ethephon application.

A number of complicating factors limit interpretation
of these data. 1) A uniform temperature was not maintained during
the absorption period. 2) Control and treated foliage were held in
the same growth chambers, leading the authors to suggest that ethylene
from the treated foliage mayhave stimulated ethylene production by the
control foliage. 3) Selection of fruit at the same physiological
state with respect to the climacteric was reportedly very difficult.

4) The treatment was replicated only two times. 5) Fruit held

I5

Table 2. Effect of temperature on rate of ethylene evolution from
control and ethephon treated tomato tissue.1

 

 

 

. QIO "Ea"
Tissue Treatment (IO-20°C) (Kcal mole-1)
Fruit Control, air 2.75 16.6

Ethephon, air 3.00 18.0
A ethephon & controlz, air 14.73 A; 44.2
Ethephon, N2 2.14 12.5
Foliage Control, air 2.08 12.0
Ethephon, air 13.87 43.2
A ethephon & control, air 15.56 45.1

Ethephon, N2 Treatment not performed

 

1Data recalculated from Lougheed and Franklin (1972).

The rate of ethylene evolution from control tissue was subtracted
from the rate for ethephon treated tissue at each temperature and
the difference was analyzed as an Arrhenius plot.

2

16

in N2 may not be comparable to those held in air. Temperature re-
sponses similar to those estimated for endogenous ethylene production
(Table I) were observed for control and ethephon treated fruit under
both N2 and air (Table 2). The ethylene evolved under N2 was assumed
to result only from ethephon degradation (Lougheed and Franklin,
1970). The difference between the ethylene evolved from control and
ethephon treated fruit in air at 10 and 20°C (0.01 and 0.62 ul Kg-1
hr-l) represents the ethylene evolution resulting from ethephon de-
gradation and any additional endogenous ethylene induced by the ethe-
phon. However this difference is actually smaller than the value ob-
served for treated fruit under N2 at 10 and 21°C (0.51 and 1.19 ul
Kg.1 hr'l) and raises the question of adequacy of the experimental
method. Unfortunately, a similar comparison cannot be made for
tomato foliage because ethephon treated foliage was not examined
under N2.

Recognizing the above limitations, some comparison of the
values in Table 2 are of interest. Because the amount of ethephon
absorbed was not controlled, probably only the relative magnitude of
the values in Table 2 is meaningful. The 010:10-20°C for endogenous
ethylene evolution from tomato fruits and foliage in air is between
2 and 3 (Table 1). This might be assumed to hold for both natural and

induced ethylene evolution since the same mechanism of synthesis has

been reported for both (Abeles and Abeles, 1972). Therefore, the

17

high 010 values reported in Table 2 must be due to ethephon degrada-
tion.

The 010 values for both control fruit and foliage were
typical for endogenous ethylene evolution (Table I). The values ob-
tained by taking the difference of the rates of ethylene evolution
for control and ethephon treated fruit in air at each temperature for
both fruit and foliage have 010 values suggesting that most of the
ethylene evolved was derived from ethephon degradation. This also
appears to be true for ethephon-treated foliage in air, but ethephon
treated fruit in air has a 010 in the range of endogenous produc-
tion. Ethephon degradation must, therefore, be contributing little
to the total ethylene evolved. This may be due to poor absorption of
the ethephon through the thick fruit cuticle, characterized as 2-6 pm
thick (Bukovac, et al., l971a). Unfortunately, ethephon treated

foliage was not studied under N so that it might be compared with

2

ethephon treated fruit under similar conditions. No explanation is
offered for the apparent contradiction of the low "Ea" for ethephon

treated fruit under N and the much greater "Ea" for the A ethephon

2
treated and control fruit in air.

18

Summary and Statement of Problem

 

Ethephon has become indispensible to the efficiency,
economy, and programing of cherry harvest. It has made mechanical
harvest of sweet cherries practical for the first time. Ethylene
is involved in fruit ripening, fruit and leaf abscission, lenticel
swelling, gummosis, and terminal dieback. Many environmental fac-
tors affect endogenous ethylene production as well as the sensi-
tivity of the tree to ethylene. Temperature affects both ethylene
biosynthesis and the degradation of ethephon t0 ethylene. Field ob-
servation has indicated that intensity of certain undesirable (e.g.,
gummosis) effects of ethephon may be associated with high tempera-
iture. Little detailed work has been done on the effect of temper-
ature on the rate of ethylene evolution from ethephon or from ethe-
phon treated tissue. This study focuses on the effect of tempera-
ture on ethylene evolution from ethephon treated leaf and shoot
tissues and from solutions of ethephon in an effort to better

understand the limitations of ethephon as an agricultural chemical.

METHODS AND MATERIALS

General Procedure

The rate of ethylene evolution from leaf and shoot
tissues and buffered ethephon solutions was studied in sealed glass
test tubes positioned vertically in controlled temperature water
baths maintained at t 0.5 degrees of the desired temperature. The
tubes were sealed with rubber serum caps at two minute intervals so
that the atmosphere could be sampled at a uniform time interval from
the start of ethylene accumulation. One ml samples of the atmosphere
in the tube were assayed for ethylene by gas chromatography using a
Packard gas chromatograph (series 7300). A103 was used as the solid
phase and N2 as the gas phase (80 cc min-1) in a glass column (120 x
0.2 cm, i.d.). The injection port temperature was maintained at
130°C, the column at 50°C, and the flame (H2 and air) ionizing de-
tector at 180°C. Data were quantified by comparing peak heights to
those of ethylene standards of known congentration. Ethylene was
measured either as total accumulation over a designated time period
or as a time course with periodic determinations over a designated
time interval. In time course studies, one ml of air was injected

19

20

into the sample tube so that the pressure in the tube would not be
affected by repeated sampling. Mean rate data for ethylene evolution
were transformed (log) to obtain homogeneous variance between treat-
ments for statistical analysis. Individual values, rather than means,

were used for all analyses of Arrhenius plots.

Experiments with Leaves of
Sour Cherry

Ethephon (125 ug) was applied as 5 droplets (5000 ppm, w/v)
of 5 ul each onto the upper surface of uniform damage-free leaves
borne on the median portion of current season's shoots of vigorous

sour cherry (Prunus cerasus L., cv. Montmorency) trees growing at

 

the Michigan State University Horticulture Research Center. This
dose was approximately equivalent to a 400 ppm dilute spray if 0.3
m1 of spray solution were retained by a leaf. The droplets were de-
livered with a 50 ul syringe along the length of the leaf within one
cm either side of the midvein and directly over secondary veins.
Generally, after a 24 hour absorption period, the shoots with
treated leaves were collected and transported to the laboratory.
Leaves with about 1.5 cm petiole attached were positioned with the
petiole in 2 m1 of water in 75 ml test tubes. Each tube contained

2 overlapping leaves with the adaxial leaf surface oriented toward

21

the outside. The average gas space in the tube was 72 ml. Tempera-
ture was maintained by holding tubes in water baths. Ethylene was
accumulated for no more than one hour at a time. If tubes were
maintained in the water baths and not sealed, a porous stainless
steel pad was inserted in the tube to just above the leaves, making
sure of good wall contact. This pad acted as a thermostat, allowing
good air exchange but preventing a temperature gradient from develop-
ing in the tube (Olien, 1971). Since treated leaves may evolve
ethylene endogenously, as well as by ethephon degradation, the rate
of ethylene evolution was corrected for leaf fresh weight and ex-

I

pressed as pmole 9’1 time- assuming the ideal gas law. The average

leaf fresh weight was 0.8 t 0.16 g.

A. Effect of temperature on rate of
ethylene evolution from ethephon
treated sour cher:y_leaves.

In the first experiment, leaves were collected 20 hours after
ethephon application and placed at 10, 18, 26, or 34°C for 4 hours.
Ethylene was accumulated over the fourth hour of incubation and the
final ethylene concentration was determined for 12 replications.

The rate of ethylene evolution was more critically determined
in a second experiment measuring accumulation periodically during the

initial hour of incubation at 18 or 33°C with 8 replications. Leaves

were collected 22 hours after ethephon application.

22

B. Effect of change in temperature
on rate of ethylene evolution
from ethephon treated leaves.

 

 

 

Leaves in the first experiment were collected from the field
20 hours after ethephon application and incubated at 10, 18, 26, or
34°C for an initial 4 hours and then transferred to 26°C for an addi-
tional 4 hours. Final ethylene accumulation was determined over the
fourth hour after transfer with 12 replications.

In a second experiment, leaves were collected 22 hours after
ethephon treatment. Ethylene evolution was determined by time course
over the initial hour of 4 sequential 2-hour time courses beginning
at 22, 24, 26, and 28 hours after ethephon application. Temperature
treatments were as follows: I) constant 18°C, 2) constant 33°C, 3)
alternating l8/33°C for periods of 2 hours, 4) alternating 33/18°C
for periods of 2 hours. The effect of temperature on ethylene evolu-
tion could then be compared over all four temperature treatments for

each time course. These treatments were replicated 4 times.

C. Effect of temperature on rate of
ethylene evolution from ethephon
treated leaves over several days.

Leaves were collected 24 hours after ethephon treatment and

held at 18 or 33°C for 70 hours. The average field temperature during

23

the 24 hour preincubation period was 20°C. The rate of ethylene evo-
lution was determined at varying intervals from 2 to 20 hours. The
rate over the initial 24 hours was estimated from the Arrhenius plot
in Figure 5, Line a. The experiment was replicated 2 times. A first
approximation of reaction order, assuming all the ethylene evolved
resulted from ethephon degradation, was determined for leaves

after initiation of the 33°C treatment, by testing goodness of linear

fit for the following plots:

 

reaction order equation
zero C = C0 - kt
first 1 In C = 1n C0 - kt
second C'] = C0-1 + kt
third c"2 = Co'2 + 2kt

These are the integral form of the equations defining reaction order.
Here C = concentration of ethephon, Co = initial concentration of

ethephon, k = rate constant, and t = time.

Effect of Temperature and Previous
Ethephon Treatment on Rate of Ethylene
Evolution from Quiescent Sweet Cherry Shoots

First year shoots were collected before bud swell on April 25,

1976 from control and ethephon treated (300 ppm at 250 gal. acre-1)

24

sweet cherry trees (Prunus avium L., cv. Napoleon) in an orchard near

 

Traverse City, Michigan used in a study on fruit abscission in July,
1975. The rate of ethylene evolution was determined for three repli-
cations of both control and ethephon treated shoots at 20, 30, and
40°C. The terminal 13 cm of the shoot was sealed in a 27 m1 test
tube for 9 hours with samples taken at l, 3, and 9 hours. The rate

of ethylene accumulation was expressed as pmoles g-l min-1.

Experiments with Ethephon Solutions

Ethephon solutions, buffered with 1 m1 of citrate (0.1 M) -
dibasic sodium phosphate (0.2 M) buffer, were prepared in 27 ml test
tubes. Buffer of the appropriate pH was equilibrated in the tubes
at the desired temperature for 10 minutes, after which 25 ul of ethe-
phon at 5000 ppm (125 pg), as was applied to leaves, was introduced
into each tube with a 50 pl syringe and the tube was sealed. The
data were collected as time course measurements, except for the

titration study.

25

A. Effect of temperature on the rate
of ethylene evolution from ethephon
solutions buffered at pH 6.0.
Solutions of ethephon buffered at pH 6.0, were monitored at
10, 20, 30, and 40°C by the methods outlined above, except that 0.17
ml of 0.01N KOH was added to neutralize the ethephon present. This
was later found to be unnecessary. The concentration of ethephon was

104.6 ppm (7.24 x 10'4

M) and the gas space over this solution was

26.1 ml. Two of nine replications were performed with sterile solu-
tions to determine the possible effect of microorganisms. In addi-
tion, one replication each was examined at pH 5.0 and 7.0. Rate of

ethylene evolution was expressed as pmole min-1 assuming the ideal

gas law.

B. Effect of temperature andppH on
the rate of ethylene evolution
from buffered ethephon solutions.

Ethephon solutions were buffered at pH 3.0, 3.5, 4.0, 4.5,
and 5.0 and held at 20, 30, 40, and 50°C. No KOH was added to the
solutions. The concentration of ethephon in each tube was 122.0 ppm

4

(8.44 x 10- M) and the gas space over this solution was 26.3 ml.

The experiment was replicated once.

26

C. Effect ofng on rate of ethylene
evolution from buffered solutions
held at 20°C.

Rate of ethylene evolution from ethephon (125 ug) solutions
buffered over a range of pH values in 75 ml tubes was examined at
20°C in two experiments. In the first experiment, 5 ml buffer was
used in each tube and the rate of ethylene evolution was determined
from the final accumulation over 43.6 hours for one replicate each
at pH 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8,
and 5.0. In the second experiment, 1 ml buffer was used in each
tube, and the rate of ethylene evolution was determined from the

final accumulation over 45.25 hours for one replicate each at pH

4.0, 5.0, 6.0, and 7.0.

D. Titration curve for ethephon.

A titration curve for 100 ml of a 0.01 N ethephon solution
titrated with 0.1N NaOH was determined with two replications. The
first replication was titrated in 0.2 ml steps. The second replica-
tion was titrated in 1.0 mlpstepsaandnrequired less time for the titra-
tion to end point. The pH was monitored with a Beckman pH meter
(Model Century SS) with a reference and a glass electrode. NaOH was
delivered from a 100 ml burette and the solution was gently agitated

with a magnetic stirrer at 22°C.

27

E. Effect of ethephon concentration
on rate of ethylene evolution.

 

Ethephon solutions of 24.4, 12.2, 2.44, 1.22, and 0.244 ppm
buffered at pH 6.0 were prepared in 1 ml buffer. The rate of ethylene
evolution was determined by time course at 20°C with one replication.
A first approximation of reaction order was then obtained by the de-
gree of linear fit for the differential form of the equations defin-

ing reaction order in terms of concentration and rate.

p§ge[_ equation

zero rate = -k[ethephon]0
first rate = -k[ethephon]1
second rate = -k[ethephon]2
third rate = -k[ethephon]3

RESULTS

Effect of Temperature on Rate of
Ethylene Evolution from Ethephon
Treated Sour Cherrpreaves

The rate of ethylene evolution increased markedly from sour
cherry leaves treated with 125 pg ethephon. The first experiment
(Table 3) indicated an "Ea" of about 30 Kcal moie'1 (010:10-20°c = 6.3).
The coefficient of determination for this linear regression was 86.2%,
but the error in the slope was only 5.9% of the slope value, indicat-
ing greater variability between leaves at a given temperature than
the response across temperatures. An analysis of the overall Arrhenius
plot for the second experiment (Table 4) gave results similar to the
first experiment, with an "Ea" of about 32 Kcal mole'1 (010:10-20°C =
7.1). The coefficient of determination was 92.4%, and the error in

the slope was 7.7% of the slope value. The results of these two ex-

periments can be compared visually in Figure 2.

28

29
Table 3. Effect of temperature on rate of ethylene evolution from
ethephon treated leaves, first experiment.1

Ethylene accumulation was determined over the fourth hour
of temperature treatment, 24 hours after ethephon

 

 

application.
Temperature (°C) Ethylene evolution (pmole 9’1 min'I)2
3
10 2.88a - 1.30
18 24.35 3 23.3
25 83.2c i 47.1
34 , 179d T 82

 

1An "Ea“ of 30.1 i 1.78 Kcal moie"1 and Q 0: 10-20°c = 5.26 was indi-
cated by the overall Arrhenius plot of 4A observations with an error
in the slope of 5.9% and r2 = 86.2%.

2Data are means of 12 replications.

3Mean separation at P = 0.05 by Duncan's Multiple Range Test on the
log transformed data.

30
Table 4. Effect of temperature on rate of ethylene evolution from
ethephon treated leaves, second experimentJ

Evolution rate was determined over a one hour time course
22 hours after ethephon application with an average r2 of

 

 

98.4%.
o . -l . -1 2
Temperature ( C) Ethylene evolution (pmole g min )
18 24.9a3 i 9.2
+
33 385b - 187

 

1An "E " of 32.1 t 2.45 Kcal mole"1 and 010:10-20°C = 7.05 was

indicated by the overall Arrhenius plot 2f 16 observations
with an error in the slope of 7.7% and r = 92.4%.

2Data are means of 8 replications.

3Mean separation at P = 0.05 by a t test on the log transformed
data.

31

FIG. 2.--Temperature effect on rate of ethylene evolution
from buffered ethephon solutions, control tissues,
and ethephon treated tissues.

Line a: Ethephon treated leaves, first experiment (Table 3)
Line b: Ethephon treated leaves, second experiment (Table 4)
Line c: Ethephon buffered at pH 6.0 (Table 9)

Line d: Endogenous production from control sweet cherry
shoot tissue (Table 8).

In Rate (In p mole min")

32

  
  
 

 

 

7-
x c 9: 57.9-16100X — Ethephon treated leaves
\ \ (r2= .989) -- Ethephon buffered at pH6
6 .. \ -"Endogenous, shoot tissue
\
\
5 .
9:58.6-15100x
4 .
3 " A
Y = 54.7-15200x
(r2=.8621

2 .

| ,_

0 F-

\. A
-1. "\.\ d Y=l4.0-4620X
\‘\. r =.905)
\ . \.
-24
-3 P 1 1 1
3.195 3.300 3.413 3.534 (°K-'x|03)
40 3o 20 10 (°C)
Tern pe roture

Figure 2

33

Effect of Change in Temperature on
Rate of Ethylene Evolution from
Ethephon Treated Leaves

The initial incubation temperature did not significantly
(P= 0.05) affect rate of ethylene evolution during the fourth hour

after transfer to 26°C (Table 5).

Table 5. Rate of ethylene evolution at 26°C from ethephon treated
leaves as affected by preliminary temperature treatment.

Leaves were collected 24 hours after ethephon application
and exposed to 10, 18, 26, and 34°C for a 4 hour period.
At the end of this time, all leaves were transferred to
26°C and final ethylene accumulation was measured over
the fourth hour after transfer.

 

 

 

Temperature treatment (°C) Ethylene evolution (pmole g;l min-1)1
10 -> 26 I 87.0a2 T 34.0
18 —> 25 ‘ 73.4a 1' 38.9
25 —4 26 72.0a T 45.9
34 —+ 25 - 55.6a i235

 

1Data are means of 12 replications.

ZMean separation at P = 0.05 by Duncan's Multiple Range Test on the
log transformed data.

34

The rate of ethylene evolution was independent of previous tempera-
ture exposure over three sequential temperature reversals between
18 and 33°C, as compared to controls at 18 and 33°C (Table 6).
Furthermore, rate of evolution changed in less than 10 minutes
following the change in temperature. All time course regressions

indicated a good linear fit (average r2 = 98.4%).

Effect of Temperature on Rate of
Ethylene Evolution from Ethephon
Treated Leaves Over Several Days

The rate of ethylene evolution from leaves held at 18°C re-
mained fairly stable throughout the experiment (Table 7). Total
estimated ethylene evolution during 94 hours accounts for only 29%
of the total possible had all the ethephon applied been degraded.
The rate of evolution from leaves held at 33°C declined with time,
falling to the range for leaves held at 18°C after 30 hours (Table
7). Total estimated ethylene evolution accounted for 46% of that
possible from the ethephon applied. The data for leaves at 33°C
fit all but the zero order reaction plots with P = 0.01. Only the
second and third order plots are linear with P = 0.001, and the

correlation coefficient was higher for the third order (r = 0.973)

than for the second order plot (r = 0.962).

35

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m

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.cowumow_qam cocamcpw 502% week?
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m_m.o w mmm¢.o mo.m w 30¢.m mo.~ w amm.m mmp.o w womm.o mm 1 cm N
Pm.m H nmk.m Rm.m H nmo.m ¢m_.o H_m¢m¢.o ¢m_o.o w mmmum.o mm 1 mm P

o.m_\.mm . . 0amm\aw_ .
mcmumccmpF< 05mm pcmpmcoo mcepmcewup< camp acmpmcou wmczoo
NAF1CPE P1m mmPoEQV gowns—o>m mamFxgum Acsvpmswp meek

 

 

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mpmmcm>mc mczpwemasmh .x¢.mm mo N; mmmcm>m cw cue: cowgmnzocw mczumgmasmp mo Lao; emcee
mg» Lm>o umpumFFou mpcmsmgammms wmczou we?» mo cowmmmgmmg ecu Eogm umcwscmuwv «L03 mmpwm

.mo>mw_ zucmgu Lzom umpmwgu
cocqmgpm seem cowpzpo>m mcmpx5em we mum; co mwgzpmgmaewp mcpumccmupw m> ucmumcou mo uummwu .m mpnmh

36

Table 7. Effect of temperature and time on ethylene evolution from
ethephon treated leaves.

Leaves collected 24 hours after field application of
ethephon and maintained after that time at 18 or 33°C.
The average temperature during the 24 hour preincubation
period was 20°C.

 

 

1

 

Time after Mean rate Total % of total
Treatment Temp.(°C) C2H4 ev.. _] C2H4 _ possible from
(hours) (pmole g-1 min ) ev. (n mole g 1) ethephon (%)
0 - 24 20 42.1 60.7 7
24 - 34 33 232 I98 23
34 - 44 33 I33 277 32
44 - 54 33 79.7 325 38
54 - 64 33 45.1 352 41
64 - 74 33 33.2 372 43
74 - 94 33 24.6 401 46
0 - 24 20 42.1 60 7 7
24 - 29 18 31.4 70 6 8
29 - 34 18 38.4 82.1 9
34 - 54 18 44.7 136 16
54.- 94 18 47.5 250 29

 

1Data are means of 2 replications.

37

Effect of Temperature and Previous
Ethephon Treatment on Rate of
Ethylene Evolution from Qpiescent
Sweet Cherry Shoots

 

 

Both control and ethephon treated shoot tissue (Table 8) had
an "E3“ in the range common for endogenous ethylene evolution (Table
1). "Ea“ was about 9 Kcal moie" (010:10-20°c = 1.75) for controls

and about 14 Kcal mole-1

(Q10:10-20°C = 2.41) for ethephon treated
shoots (Table 8, Figure 2). These values were significantly differ-

ent from each other at P = 0.025.

Effect of Temperature on the Rate of
Ethylene Evolution from Ethephon
Solutions Buffered at pH 6.0

The effect of temperature on ethylene evolution from ethephon
buffered at pH 6.0 was similar to that observed for leaves treated
with the same dose (Table 9, Figure 2). No deviation was observed
with the sterile solutions from the nonsterilized replications. The

overall “Ea" was 32 Kcal mole-1

(Q10:10-20°C - 7.0). The coefficient
of determination for this regression was 98.9% and the error in the
slope was 1.8% of the slope value. The Arrhenius slopes for the
single replication at pH 5.0 and pH 7.0 were not significantly dif-

ferent from each other or from the overall Arrhenius slope for pH 6.0

even at P = 0.50.

38

Table 8. Effect of temperature on rate of ethylene evolution from
'Napoleon' sweet cherry shoot tissue collected before
bud swell.

Napoleon sweet cherry trees sprayed dilute with 300 ppm
ethephon in July, 1975. Quiescent control and treated
first year shoot tissue collected April, 1976.

 

 

 

Temperature (°C) Ethylene evolution (pmole g.1 min-1)1
0 ppm2 300 ppm3
20 0.1551 T 0.002 0.124 1 0.030
30 0.305 - 0.025 0.358 T 0.031
40 0.433 f 0.093 0.591 T 0.090

 

1Data are means of 3 replications.

2An “E " of 9.18 i 1.13 Kcal moie'1 and Q :10-20°c = 1.75 was

indicated by the overall regression for control tissue of 9
observations with an error in the slope of 12.3% and r2 = 90.5%.

3An “E " of 14.4 f 1.59 Kcal moie" and 0 :10-20°c = 2.41 was indi-

cated by the overall regression for treatgd tissue of 9 observations
with an error in the slope of 11.0% and r = 92.1%.

Table 9.

39

Effect of temperature on rate of etherne evolution from

ethephon solutions buffered at pH 6.0.

Rate of evolution from 7.24 x 10"4 M ethephon solutions
(125 pg ethephon) buffered at pH 6.0 determined by time

course with an average r2 of 98.9%.

 

Temperature (°C)

Ethylene evolution (pmole min-1)2

 

10

20

30

40

3.85a3

16.5b
l32c

817d

l+

+

l+

I+

0.73

2.07

6

68

 

I

in the slope of 1.8% and r2 = 98.9%.

zData are means of 9 replications.

An "E “ of 31.9 T 0.58 Kcal moie"1 and 0 :10-20°c = 5.99 was indi-

catedaby the overall Arrhenius plot of 3Soobservations with an error

3Mean separation at P = 0.05 by Duncan's Multiple Range Test on the
log transformed data.

40

The Effect of Temperature and pH on
the Rate of Ethylene Evolution from
Buffered Ethephon Solutions

 

 

The energy of activation does not appear to be affected by
pH. The Arrhenius slopes for pH 3.0, 3.5, 4.0, 4.5, and 5.0 were not
significantly different even at P = 0.20 (Table 10). Furthermore,
the slope of the Arrhenius plot over all five pH values ("Ea" = 35
Kcal mole-T. Q10 = 8.5) was not significantly different from the

overall Arrhenius slope for pH 6.0 at P = 0.50.

Effect of_pH on Rate of Ethylene
Evolution from Buffered Ethephon
Solutions Held at 20°C.

The rate of ethylene evolution from buffered ethephon solu-
tions increases exponentially with increase in pH (Figure 3).

Degradation of ethephon at 20°C was not detectable below pH 3.0.

Titration Curve for Ethephon

Two pKa's are evident for ethephon (Figure 4) at about pH
2.5 and 7.0. The end point for the titration with the glass elec-
trode used was at pH 11. The pka's for phosphorous acid are at pH

2.15 and 4.7 (25°C), and for phosphoric acid are at pH 1.96, 7.12,

41

Table 10. Effect of temperature and pH on the rate of efihylene
evolution from buffered ethephon solutions.]’

Rate of evolution from 8.44 x 10’4 M buffered ethephon
solutions (125 pg ethephon) determined by time course
with an average r2 of 99.4%.

 

. . -1 3
Temperature (°C) Ethylene evolution (pmole min )

 

pH 3.0 pH 3.5 pH 4.0 pH 4.5 pH 5.0

 

20 .02111 .0592 .0172 .536 1.80
30 .179 .448 1.46 3.81 13.1
40 .875 2.34 7.18 23.2 72.7
50 8.32 15.2 52.0 127 520

 

1An "E “ of 35.0 t 6.07 Kcal mole-1 and Q :10-20°C = 8.45 was indi-

catedaby the overall Arrhenius plot of ZOoobservations with an error
in the slope of 17.3% and r2 = 54.9%.

2The Arrhenius slopes for each pH are not significantly different at
P = 0.20.

3Data are of one replication.

42

and 12.32 (18°C) (The Handbook of Physics and Chemistry, 1953). The
rate of ethylene evolution does not show a dependence on proton dis-

sociation from ethephon (Figures 3, 4).

The Effect of Ethephon Concentration
on Rate of Ethylene Evolution

The rate of ethylene evolution from ethephon solutions
buffered at pH 6.0 increased with ethephon concentration (Table 11).
The data significantly fit a linear plot for a first order reaction
at P = 0.001, for a second order reaction at P = 0.01, and for a
third order reaction at P = 0.05. The linear correlation for a

zero order reaction plot was not significant at P = 0.50.

Table 11. Effect of ethephon concentration on rate of ethylene
evolution.

Ethephon buffered at pH 6.0 and held at 20°C Rates
determined by time courses with an average r2 of 91.5%.
Preliminary experiment consisting of one replication.

 

 

Concentration (ppm) Ethylene evolution (pmole min'I)
24.4 2.24
12.2 . 1.03
2.44 0.211
1.22 0.0936

0.244 0.0399

 

43

FIG. 3.--The effect of pH on rate of ethylene evolution
from buffered solutions of ethephon held at 20°C.

44

~15C)

AI ._-.=._ .5 20m

~ICMD

i 5C)

 

P

p F

3 2

7.1.7.... .5 03m

0.

 

Figure 3

45

FIG. 4.--Titration cur
ve for 100
with 0.1N NaOH. ml 0'01” ethephon titrated

46

 

b

L5-

32: 1052

10

 

U
0 5 0

Figure 46

DISCUSSION

The effect of temperature on rate of ethylene evolution from
ethephon and from ethephon treated sour cherry leaves was much greater
than expected. It is a general rule for a reaction with activation
limits that the rate of reaction approximately doubles for a 10°C rise
in temperature (Q.l0 = 2). A Q10 of 2 calculated between 10 and 20°C

is equivalent to an Ea of 11.4 Kcal mole-1.

Ethephon solutions and
ethephon treated leaves evolved ethylene with an "Ea" of about 30 Kcal
mole'1 (Figure 2), while nontreated sweet cherry shoot tissue collected
before bud swell (Figure 2) evolved ethylene with an "Ea" of 9.2 Kcal
mole-1. Published data indicate that endogenous ethylene was evolved
within the expected Ea range regardless of species or tissue (Table 1).
Why the rate of ethylene evolution from buffered ethephon solutions and
from ethephon treated leaves is so responsive to temperature is not ex-
plained by this study. The response is not due to pH differences
(Table 10) nor to differential uptake of the compound by leaves. A

defined dose of ethephon was applied to each leaf and was followed

with an absorption period in the field.

47

48

With the present data, we can predict what the limits on
"Ea" might be for ethephon treated tissue (Figure 5). The ethylene
evolved from ethephon treated tissue may arise from degradation of
ethephon, endogenous biosynthesis, or both. If all the ethylene
were endogenously produced, we could expect an “Ea" of 14.6 i 4.8
Kcal mole”1 (Figure 5, line b). If all of the ethylene were derived
from ethephon degradation, we could expect an "Ea“ of 31.6 T 0.9 Kcal
mole-1 (Figure 5, line a). If half the ethylene came from one source
and half from the other, the "Ea" would be expected to fall half way
between these limits, at 23 Kcal mole-]. The Arrhenius slopes of the
data from two leaf experiments (Tables 3, 4), the experiment with
ethephon buffered at pH 6.0 (Table 9), and the experiment examining
the effect of pH on Ea (Table 10) were found not to differ signifi-
cantly. Therefore, essentially all of the ethylene evolved from
leaves treated with ethephon, in the manner specified, must be de-
rived from the degradation of ethephon. This conclusion is supported
by the estimated values for total ethylene evolution during 94 hours
from ethephon treated leaves (Table 7). The total ethylene accumu-
lated after 24 hours at 20°C and an additional 70 hours at 33°C
accounted for only 46% of the total ethylene possible if 100% of the
ethephon applied had degraded. The total ethylene accumulated after
24 hours at 20°C and an additional 70 hours at 18°C accounted for

only 29% of the total possible.

49

FIG. 5.--Estimated limits on the Arrhenius slope for rate of

Line a:

Line b:

ethylene evolution from ethephon treated tissue.

Ethephon degradation (estimate of upper limit on
Arrhenius slope for ethephon treated tissue).
Overall regression of data from Tables 3, 4, 9.
Y = 50.5 - 15900x r2 = 0.920

Endogenous ethylene evolution (estimate of lower
limit on Arrhenius slope for ethephon treated
tissue).

From Table 1.

Slope = 7350 T 2420 deg

Intercept with line a is arbitrary.

In Rate (In pmole min")

 

50

 

7.
6r»
5..
4 _ Ethephon Degradation
3.
b
24 Endogenous Ethylene Evolution
1.
3.195 3.300 3.413 3.534 (°K"x 1031
40 30 20 IO (°C)
Temperature

Figure 5

51

The opposite situation to that of the treated leaves might
explain the data for control and treated sweet cherry shoots (Table 9)
collected 9 months after ethephon treatment. The "Ea“ for control
shoots was about 9 Kcal mole—1 and the “E3" for the treated shoots
was about 14 Kcal mole-1, and the slopes were significantly different
at P = 0.025. These data suggest that enough ethephon remained to
affect the ethylene evolution response to temperature.

The best estimate of the upper limit for the "Ea“ of ethephon
treated tissue (Figure 5, line a) was obtained by combining the data
from the two leaf experiments (Tables 3, 4) and the experiment with
ethephon buffered at pH 6.0 (Table 9). The experiment examining the
effect of pH on Ea (Table 10) was excluded because of excessive error.
The overall regression of these 100 observations gives an "Ea" of
31.5 i 0.9 Kcal moie‘1 (010:10-20°c = 5.9) with a r2 of 92.0%.

The best-e§timate of the lower limit on "Ea" (Figure 5, line
b) for ethephon treated tissue where all the ethylene evolved is en-
dogenously produced was derived from the recalculated rate data sur-
veyed from the literature (Table I). Some of this is not given in
units that can be converted to pmole g.1 min-1. For this reason the

plot was arbitrarily made to pass through the same point at 10°C as

 

was determined for line a, to aid in visually comparing slopes.
The rate of ethylene evolution from ethephon treated leaves

shifted rapidly and reversibly with change in temperature. The first

52

experiment (Table 5) indicated that the effect of temperature was
reversible, but not how fast or how often. A second experiment
(Table 6) demonstrated that the effect of temperature was completely
reversible over several temperature reversals between 18 and 33°C.
The change in rate occurred within 10 minutes. The r2 values calcu-
lated after each temperature reversal were very high and similar to
the r2 values for the control leaves (Table 6).

The rate of ethephon degradation increases exponentially
with increase in pH (Figure 3). This increase is independent of
the regions of proton dissociation apparent from the titration
curve for ethephon (Figure 4). This would indicate that, at least
at pH 3.0 and above, rate is not controlled by the degree of proton
dissociation. Degradation of ethephon at 20°C was not detectable
below pH 3.0.

A first approximation of reaction order for the degradation
of ethephon indicated a first order reaction in solution but some
higher order reaction when applied to leaves. The data, especially
that from leaves, are preliminary, but these results could be ex-
plained by additional factors becoming limiting in leaves. Limiting
factors in leaves but not in solution might be availability of the

nucleophyllic attacker referred to by Yang (1969) or the binding of

ethephon with plant components. There is evidence that ethephon can

53

bind to sucrose (Lavee and Martin, 1971) and with sweet cherry fruit
cell wall components (Edgerton and Hatch, 1972). Whether or not the
reaction order for ethephon degradation differs between solutions
and treated leaves, the effect of temperature on ethylene evolution
has been shown to be the same in both cases for a dose of 125 pg

ethephon.

CONCLUSIONS

The rate of ethephon degradation increases markedly with tem-
perature for leaves treated with 125 pg ethephon and for solutions of
ethephon at pH.3 and above. This temperature dependence can be ex-
pressed quantitatively by an "Ea“ of about 30 Kcal mole"1 (Q10:10-20°C
= 6.9). Why this value is so high is not known, but the fact itself
is very important to the use of ethephon in agriculture. The degree
that the rate of ethylene evolution is affected by temperature must
depend on the ratio between ethylene derived from ethephon degradation
and that produced endogenously. This ratio will depend on many fac-
tors, including the dose of ethephon applied and the capacity of the
tissue for biosynthesis of ethylene. Absorption of ethephon may also
be an important factor, and this is probably controlled by tempera-
ture, moisture, cuticle characteristics, etc. For these reasons,
the temperature at the time of ethephon application in the orchard
and for several days afterward is an important factor in determining
response. This may, in part, explain the widespread ethephon induced
phytotoxicity which was associated with the high temperatures of the

1973 cherry harvest season.

54

55

In many cases, the temperature effect on ethephon treated
tissue does not appear to limit the usefulness of the compound. In
cases where temperature may limit utility, the does applied must be
adjusted in accordance with the predicted temperature over some num-
ber of days from ethephon application. Temperatures that are too
low or too high may prevent the use of ethephon. Although not econ-
omically practical at this time, a potential method of avoiding the
effects of high temperatures on sour cherry might be the use of
evaporative cooling. This should be effective because of the rapid
decline in the rate of ethylene evolution from ethephon treated
tissue with a decline in temperature. Where temperature is a chronic
problem, the use of some other existing compound or the development
of a new one might be necessary, so that the effect of temperature

on treated tissue is not so great.

APPENDICES

APPENDIX A

THE ARRHENIUS EQUATION

For an isothermal reversible reaction that involves no radiant

or electrical work, the change in Gibbs free energy is expressed by:

AG = AH - TAS G = Gibbs free energy
H = enthalpy
T = absolute temperature
S = entropy

The standard Gibbs free energy is defined by the activity of
the reactants being equal to the activity of the product, so:

AG = AG° = -RT an G° standard Gibbs free energy

 

R ideal gas constant
K = activity ofpproducts
activity of reactants
_ eves
and an RT R

Over temperature intervals that are small in terms of absolute temper-
ature, H and S are assumed to be constant. This approximation can
generally be made for reactions involving no electrical or radiant
work terms and not involving phase changes. When the last equation

is differentiated with respect to temperature, we obtain:

 

57

This equation can be partitioned into the forward and reverse reac-

 

 

tions.
AH = E f - E r E = activation energy for the
a a a .
forward or reverse reaction
Ink = In E;- k = rate constant for the for-
ward or reverse reaction
dlnkf-dlnkr=_E_a_‘: '59.:
d1 dT RTZ RTZ
d1n1<=E_a_
dT RT2

Integrating over indefinite temperature limits gives the Arrhenius

equation.

E

an = I - _§_T-1 I = integration constant
R

Arrhenius arrived at this equation empirically. Glasstone (1946)
states that, "The Arrhenius equation is widely applicable not only
to homogenous gas reactions, but also to reactions in solution and
to heterogeneous processes. It frequently fails, however, for

chain reactions."

i530

APPENDIX B

DETERMINATION OF ETHEPHON DOSE PER LEAF
FOR ADEQUATE ETHYLENE EVOLUTION

A. Procedure

Five (H2 20 drops of ethephon solutions (0, 1000, or 5000 ppm)
were applied 'to leaves in the field. Nontreated leaves were also in-
cluded in the test. Five leaf samples were collected 25 hours after
treatment and ethylene was accumulated in 265 ml jars fitted with

serum caps. The experiment was repeated two times.

B. Results

1

Chemical (ppm) Drops per Leaf Mean Ethylene Evolution (n1 9' )

 

 

 

no treatment 0 0.747
01 5 0.853
01 20 1.02
1000 5 6.62
1000 20 12.5
5000 5 40.1
5000 20 421.
1
Rep I only.

C. Conclusions
Ethephon will be applied as a 5000 ppm solution in 5 drops
(125 pg) per leaf. This is a convenient dose to apply and gives

58

59

an easily measured response at 24 hours. This dose is equivalent

to a 400 ppm dilute spray if 0.3 m1 is retained by the leaf.

APPENDIX C

ETHEPHON LEAF PHYTOTOXICITY

A. Procedure
Five drops of 0, 1000, 5000, and 10000 ppm ethephon, and pure

8 ppm) Ethrel (Amchem Products, Ambler, Penn.) were placed

(2.40 x 10
on detached sour cherry leaves arranged on the lab bench. Each treat-
ment was replicated with five leaves. Observations for leaf injury

were made over several days..

B Results
No caustic effects were noted until the second day. Severe
phytotoxicity was noted only for pure Ethrel, and none at the other

treatments.
C. Conclusions

Five drops of a 5000 ppm ethephon solution should not be

caustic to sour cherry leaves.

60

APPENDIX D

DETERMINATION OF THE AMOUNT OF ETHYLENE SOLUBLE IN
THE WATER PRESENT DURING ETHYLENE ACCUMULATION

Abeles (1973) gives the absorption coefficient of ethylene at
1 atm in water at 25°C as a = 0.108 (v/v).

Ethylene was accumulated in tubes containing 2 ml of water and
two leaves with an average combined weight of 1.6 9. Assuming these
leaves are 75% water, then we have a total of 3.2 ml water present.
This means that at 25°C, 0.3456 m1 of ethylene at 1 atm would be solu-
ble in the water present. Assuming the ideal gas law, this is equal
to 1.413 x 10.5 moles ethylene.

A concentration of 0.1 ppm ethylene is near the lower range of
what can be measured. The partial pressure of ethylene at this con-
centration is 10"7 atm in a tube with total pressure of 1 atm. At

‘12 males are soluble in the water present at

10

this pressure, 1.413 x 10
25°C. There are 2.945 x 10' moles of ethylene present at 25°C and
0.1 ppm in a gas space of 72 ml.

This means that for a tube with 3.2 m1 of water and 72 ml gas
space at 25°C, 1 atm total pressure, and 0.1 ppm ethylene, 0.48% of

the ethylene is soluble in the water present. For this reason,

61

62

solubility of the gas can be considered a negligible error in the

ethylene evolution studies made with this system.

APPENDIX E

RELATIVE SENSITIVITY 0F QUIESCENT SHOOT TISSUE
T0 ETHEPHON AMONG SEVERAL TREE FRUIT SPECIES

A. Procedure
First year wood was collected at the MSU Horticulture Farm

from apple (Malus domestica Bork., cv. McIntosh), sour cherry (Prunus

 

cerasus L., cv. Montmorency). sweet cherry (Prunus avium L., cv.

 

Windsor), plum (Prunus domestica L., cv. Stanley), and peach (Prunus

 

persica L., cv. Redhaven). Napoleon sweet cherry twigs from Oregon
were also available. These twigs were more vigorous than those col-
lected at the MSU Hort Farm. The terminal 23 cm were placed in 50 ml
solutions of 0, 50, 100, and 200 ppm ethephon on April 10, 1975.
Visual gumming and bud expansion ratings were made 7 days later and

at termination of the experiment (at 11 days). Also at termination,
browning of the wood was rated, increase in mean bud fresh weight over
the initial mean bud weight was determined, and the nI/g ethylene
accumulated over one hour for the terminal 13 cm was determined at

room temperature. All treatments were replicated three times.

63

64

B. Results

Peach was extremely sensitive to ethephon. All peach shoots
except the control were killed very shortly after being placed in any
concentration of ethephon and became dehydrated. They did not gum
and showed little internal browning. No bud expansion or increase
in bud weight occurred except in the controls. Ethylene was evolved
at the control level at all concentrations of ethephon.

Napoleon sweet cherry showed a more marked inhibition of bud
expansion, increase in bud fresh weight, and browning than any of
the other tree fruit wood tested. Less gumming was observed, however,
than occurred for either Windsor sweet cherry or Stanley plum.

The remaining species having similar vigor and remaining alive
through the experiment can be described together. Apple, Windsor
sweet cherry, sour cherry, and plum all showed similar inhibition of
bud weight and expansion with an increase in ethephon concentration.
Little additional effect was observed above 50 ppm. Gumming was most
severe in plum, less so in Windsor sweet cherry, and absent in sour
cherry and apple. No gumming was observed below 100 ppm.

The ethylene accumulation data were most useful for ranking the
varieties in sensitivity to ethephon. Generally, these fell into two
groups. The sweet cherries and plum were very responsive to ethephon.

The response began to fall off above 100 ppm. Sour cherry, peach, and

65

apple were relatively unresponsive. Peach was killed at 50 ppm and
so was not included in this comparison. The slopes of the ethephon
concentration vs. ethylene evolution plots were determined and com-
pared. Schematically, the results of this comparison can be shown
as follows:

(Apple) (Sour Cherry) (Win. Sw. Ch.) (Plum) (Nap. Sw. Ch.)
The lines indicate slopes that were not significantly different at

P = 0.05.

C. Conclusions

Summarizing all of the above observations, the five tree fruit
species tested might be ranked from lowest to highest ethephon sensi-
tivity as follows: McIntosh apple, Montmorency sour cherry, Windsor
sweet cherry, Stanley plum, and Redhaven peach. It is not known where
Napoleon sweet cherry would fall if it had been collected from the

same area as the other wood.

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