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This is to certify that the

thesis entitled

Influences of Shading and Ethylene-Releasing Compounds of

Anthocyanin Biosynthesis and Maturation of Early Apple

Cul tivars presented by

Anne Michele Swindeman

has been accepted towards fulfillment
of the requirements for

Masters Horticulture

 

 

degree in

David R. Dilley A

{ mworemé: ¢97
/

8/6/86 Graduate Enrollment Offiéér

Dept. of Horticulture

 

Date

 

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

MSU

LlBRARlES
4-42:—

v

 

 

RETURNING MATERIALS:
Place in book drop to
remove this checkout from
your record. FINES will
be charged if book is
returned after the date
stamped below.

 

 

 

 

 

 

INFLUENCES OF SHADING AND ETHYLENE-RELEASING
COMPOUNDS ON ANTHOCYANIN BIOSYNTHESIS AND MATURATION
OF EARLY APPLE CULTIVARS

BY

Anne Michele Swindeman

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1986

ABSTRACT
INFLUENCES OF SHADING AND ETHYLENE-RELEASING

COMPOUNDS ON ANTHOCYANIN BIOSYNTHESIS AND MATURATION
OF EARLY APPLE CULTIVARS

BY

Anne Michele Swindeman

High temperatures and shading reduce anthocyanin
biosynthesis in apple: fruits. Ethephon (2-
chloroethylphosphonic acid) enhances fruit coloration, but
generally initiates ripening and shortens the shelf life of
apples. The influences of shading levels and ethylene-
releasing compounds on anthocyanin biosynthesis were
investigated using early maturing apple cultivars.

‘Paulared' apple trees previously treated with low
concentrations of 2-chloroethylmethyl-bds-(phenylmethoxy)-
silane (CGA 15281), were shaded at 0%, 51%, 73% or 92% of
full sunlight.two weeks before harvest. Fruit coloration
and maturation were not affected by 0.5 mM or less CGA
15281. Shaded fruits were firmer but poorly colored and
smaller than fully exposed fruit. Fruit maturation was not
affected by shading.

‘McIntosh' apple trees were treated with ethephon or
CGA 15281 two or three weeks before harvest. Solvolytic
fragmentation of ethephon was slow and constant, resulting
in increased anthocyanin biosynthesis, autocatalytic
ethylene production and flesh softening within six days of

application. Solwnolytic fragmentation of CGA 15281

occurred quickly, especially when applied three weeks before
harvest. CGA 15281 at 1.0 mM increased anthocyanin

biosynthesis without concomitant fruit ripening.

ACKNOWLEDGEMENTS

I would like to express my appreciation to Dr. David
Dilley for the guidance, patience and friendship he offered
me while I was his student. I am especially grateful to
Dave for giving me the opportunity to pursue my own research
interests and for making sure I was never unchallenged in my
work.

Many thanks also go to my committee members, Drs. Jim
Flore and Hans Kende, who put alot of time and effort into
the planning and critiquing of my research and thesis.

Lastly, the quantity and quality of work that went into
this thesis is a reflection in part of the excellent
technical assistance of Eric Ayers, Mary Belfast and many of
my fellow postharvest graduate students who were there when

the going got tough.

iv

Guidance Committee:

The paper format adopted for this thesis is in
accordance with departmental and university regulations.
Section II and Section III were written for publication in
the Journal_ f the American Society for Horticultural

Science.

TABLE OF CONTENTS

LIST OF FIGURESO...OO...O...O...OOOOOOOOOOOOOOOOOOOOOO Viii
INTRODUCTIONOOOCOOCOCCOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 1

SECTION I. LITERATURE REVIEW.............................
Mechanics of Anthocyanin Biosynthesis................
Factors Affecting Anthocyanin Biosynthesis in

Apple Fruits........................................
Light...........................................
Ethylene........................................
Temperature.....................................
Cultivar........................................
Growth Regulators...............................

Objectives of Present Study.........................
Literature Cited....................................

#00

NI—uoxoooonn-m.

l-‘l-‘

SECTION II. EFFECT OF SHADING AND LOW ETHYLENE
CONCENTRATIONS ON ANTHOCYANIN BIOSYNTHESIS OF
‘ PAULARED' APPLES 18
Abstract............................................ 19
Materials and Methods............................... 26
Experimental design............................ 20
Harvest date prediction........................ 20
Chemical treatment............................. 21
Shading treatment.............................. 21
Quality and maturity evaluations............... 22
Statistical analysis........................... 23
Results and Discussion.............................. 23
Chemical treatment............................. 23
Shading treatment.............................. 24
Literature Cited.................................... 29

SECTION III. ANTHOCYANIN BIOSYNTHESIS AND MATURITY
OF ‘MCINTOSH' APPLES AS INFLUENCED BY ETHYLENE-
RELEASING COMPOUNDS.................................... 32
Abstract............................................ 33
Materials and Methods............................... 35
Solvolytic fragmentation of ethylene- and
propylene generating compounds................ 35
Field study.................................... 36
Harvest date prediction........................ 36
Chemical treatment............................. 37
Solvolytic fragmentation of CGA 15281 and
ethephon in apples............................ 38
Quality and maturity evaluations............... 38
V1

Results and Discussion.............................. 39
Solvolytic fragmentation of ethylene- and
propylene generating compounds 13 vitrouuu" 39
Solvolytic fragmentation of CGA 15281 and
ethephon in apples............................ 42
Quality and maturity evaluations............... 45
Literature Cited.................................... 58

 

vii

LIST OF FIGURES

SECTION II.

Figure 1. Fruit weight 0, 5, 8 and 11 days after
shading (n=l44 except at day 0 where n=960); lsd .05 =
6.80 for comparing within same sampling time, and 5.87
within same shade level.................................

Figure 2. Flesh firmness of ‘Paularedl apples 0, 5,
8 and 11 days after shading (n=72 except at day 0
where n=480); lsd .05 = 2.00 for comparing within the
same sampling time, and 1.88 within same shadelevel...

Figure 3. Absorbance at 530 nm of anthocyanin
extracted from ‘Paulared' apples 0, 5, 8 and 11
days after shading (n=l44 except at day 0 where
n=480); lsd .05 = .079 for comparing within same
sampling time, and .076 within same shade level.........

Figure 4. Visual estimation of percent surface

coloration of ‘Paulared' apples 0, 5, 8 and 11
days after shading (n=l44 except at day 0 where
n=480); lsd .05 = 4.4 for comparing within same

sampling time, and 4.3 within same shade 1evel.........

SECTION III.

Figure 1. Solvolytic fragmentation of A) 2-chloro-
propylphosphonic acid, B) CGA 15281, and C) ethephon
in citrate-phosphate buffers adjusted to pH 3, 4, 5,

6 and 7. Incubation was at 20 C (n=6).................

Figure 2. Solvolytic fragmentation to ethylene of CGA
15281 and ethephon applied to mature ‘McIntosh' trees
on A) 29 August 1984 or B) 6 September 1984.
Solvolytic fragmentation was measured as internal
fruit ethylene by gas chromatography (n=25)u.u.n.u..

Figure 3. Percent red coloration of ‘McIntosh' apples
treated with CGA 15281 or ethephon on A) 29 August
1984 or B) 6 September 1984. Red surface coloration
was estimated visually to the nearest 10% (n=25); lsd
.05 within sampling date = 12.939 (A) and 13.450 (B),
and withinconcentration= 11.260 (A)and13.l33 (B)"..

viii

26

27

28

40

43

47

Figure 4. Absorbance of anthocyanin extracted from
‘McIntosh' apples treated with CGA 15281 or ethephon

on A) 29 August 1984 or B) 6 September 1984 (n=25);

lsd .05 within sampling date = .126 (A) and .145 (B),

and within concentration=.ll9(A)and.136 (B)un.n.. 49

Figure 5. Flesh firmness of ‘McIntosh' apples at each
sampling date treated with CGA 15281 or ethephon on A)
29 August 1984 or B) 6 September 1984 (n=25);
lsd .05 within sampling date = 3.893 (A) and 3.060
(B), and within concentration = 3.597 (A) and 3.169

(B)oooooooooooooooooocoo-0000.00.00...oooooooooooooooooo 51

Figure 6. Flesh firmness at 0, 1, and 2 weeks of
‘McIntosh' apples harvested 9 days after treatment on
A)29 August 1984 or B) 6 September 1984. Fruit was
stored at 20 C (n=25); lsd .05 within sampling week =
3.737 (A) and 2.559 (B), and within concentration =
3.682(A) and2.199 (B). 54
Figure 7. Starch index of ‘McIntosh' apples treated
with CGA 15281 or ethephon on)“ 29 August 1984 or
B) 6 September 1984 (n=25); lsd .05 within sampling
date = .853 (A) and .840 (B), and within concentration
= .763(A) and .796

(B)ooooooooooooooooooooooooooooooooo 56

ix

I NTRODUCT ION

High quality, early season apple cultivars like
‘Paulared' and ‘McIntosh' potentially command high market
returns. Since most Michigan fruit is marketed as U. S.
Extra Fancy, a fifty percent red color requirement must be
met. In poor coloring years, a significant portion of the
croplmay not meet minimum grade requirements for color.
Grower returns are substantially reduced as a result.

Apple producers take several approachestx>improving
coloration. Dormant pruning, followed by summer pruning,
improves light penetration through the trees' canopies.
Mineral nutrition, i.e., nitrogen and potassium, is
monitored and adjusted accordingly. Growth regulators,
mainly daminozide (Alar) and ethephon, are used extensively.
Consistently poor coloring strains may be replanted or top
worked with high coloring strains. Some growers delay
harvest until fruit meets minimum color standards.

Although such approaches improve color significantly,
the postharvest integrity of the fruit is sometimes
adversely affected. Internal fruit ethylene levels rise
when ethephon is tunai or harvest is delayed. Consequently,
this fruit softens rapidly and is suitable only for short
term storage. If the fruit is not sold promptly, consumer

dissatisfaction, hence, wholesale buyer resistance, occurs.

1

2
Further reductions in grower returns may result not only for

these varieties, but for later season varieties as well. A
better understanding of anthocyanin biosynthesis in apples
is necessary to prevent quality problems resulting from

these chemical and cultural practices.

SECTION I.

LITERATURE REVIEW

4
Mechanics g£_Anthocyanin Biosynthesis

 

 

The anthocyanin pigment found in Malus sylvestris cv.

 

‘Jonathan' and ‘Stayman' is 3—B-galactosidylcyanidin, or
idaein (49). Idaein develops late in the growing season,
concomitant with other metabolic changes. Anthocyanin
development in ‘McIntosh' apples is correlated to an
increase in metabolic activity of the pentose-phosphate
pathway (21L.By inhibiting glycolysis and diverting glucose
metabolism to the pentose-phosphate pathway, anthocyanin
biosynthesis increases. Faust (21) reports that
anthocyanin development in apple is enhanced under these
conditions because the amount of shikimic acid is increased.

Anthocyanins are derived from shikimic acid, the direct
precursor of benzene ring B of idaein (21). The conversion
of shikimic acid to cinnamic acid requires the intermediate
formation of l-phenylalanine. Phenylalanine ammonia-lyase
(PAL) catalyzes the deamination of l-phenylalanine to trans-
cinnamic acid, thus diverting the amino acid from protein
synthesis to the phenylpropanoid pathway (31). The
hydroxylation of trans-cinnamic acid to p-coumaric acid is
next catalyzed by cinnamic acid hydroxylase or CAH (26).
Ring A is the condensation product of three acetic acid
units (24), which are attached one by one to ring B (3).

Lastly, galactose is attached to the cyanidin molecule (3).

Factors Affecting Anthocyanin Biosynthesis 1g Apple Fruits

 

Light In apple skin, anthocyanin biosynthesis has two

distinct radiation dependent phases (54). The first is a 12

5
to 20 hour induction period during which rm) visible

anthocyanin development occurs (40, 54). A linear increase
in anthocyanin follows which is a function of irradation
time at 600 to 750 nm (54). Faragher and Chalmers (20)
report similar kinetics, and in agreement with others (12,
14, 17, 25), correlate light induction or activation of the
PAL enzyme to anthocyanin biosynthesis in plant tissues.

2_ 2219 synthesis of PAL, the rate-limiting enzyme

involved in anthocyanin biosynthesis, has been observed

using radioactive labelling techniques in Xanthium (61),

 

mustard (50), and potato tuber discs (48) during light

stimulation. In Xanthium, however, the "induction" of PAL

 

is due to a decrease in the rate of PAL inactivation (62).
Camm and Towers (5) suggest that there is a continuous
production of a PAL-inactivator and that changes in PAL
levels may reflect changes in the rate of PAL inactivator
synthesis, not an increase in 92.2222 synthesis of PAL.
Zucker (59) and Engelsma (16) report that cycloheximide
(CHI) will inhibit both the appearance and disappearance of
PAL in potato tuber discs and gherkin seedlings, depending
on specific conditions. Zucker (60) reports that PAL
synthesis can be inhibited by 50% in potato tuber discs
cultured on 5 uM CHI and by almost 100% in discs cultured on
10 uM CHI. Where the maximal level of PAL is allowed to
form in discs cultured on water, i.e”, after 24 hours of
light induction, PAL disappearance is prevented by a 70 uM
CHI treatment. The loss of PAL under dark conditions also

requires d3 novo synthesis. From this work, it is suggested

6
that 1) early phases of PAL induction involve d noyg

synthesis of the enzyme in the absence of any turnover; 2) a
system which degrades or inactivates the lyase subsequently
forms in the tissue; and 3) the formation of this degrading
or inactivation system also requires d3 £219 protein
synthesis (60).

Faragher and Chalmers (20) confirm the light
requirement in apple skin of whole fruit. ‘Without light, no
anthocyanin and virtually no PAL form. Light treatments
which increase or decrease PAL levels cause comparable
changes in the rate of anthocyanin accumulation. This
indicates that PAL is the rate-limiting enzyme involved in
anthocyanin biosynthesis in whole apple fruits (20).

Apple skin discs held under dark conditions develop PAL
activity but without anthocyanin biosynthesis (20, 55).
PAL, therefore, is not considered the factor responsible for
the light requirement in apple fruits. Light may be
required for the production of some other enzyme or factor
involved. Once this light requirement is met, PAL activity
appears to regulate anthocyanin biosynthesis in whole
fruits.

Ethylene Once the light requirement.i51met,i1:is likely

 

that ethylene controls PAL activity (8). Ethylene and
treatments inducing ethylene, iAL, wounding, u.v. light,
cycloheximide, gamma irradiation and disease increase d3
ggyg_PAL synthesis in several plant tissues (7,Sh.21, 27,
28, 36, 43, 44). Some tissues known to be unresponsive to

white light treatments are responsive to ethylene treatments

(43, 44).

In studies (ME pea seedling response to ethylene
treatments, Hyodo and Yang observed a marked increase in
PAL in epicotyl segments exposed1u>10 ul 1"1 ethylene or
more (ZTL. The induction period of PAL is estimated at 6
hours with maximal activity at 30 hours. If ethylene is
withdrawn, PAL increases for a short time, then stops.
Upon reintroduction of ethylene, PAL increases. Early
additions of cycloheximide or actinomycin-D almost
completely inhibit PAL synthesis; however, when they are
added late, they stimulate PAL synthesis. This is
consistent with the view that PAL and PAL-inactivator
systems exist, and that both may require 92.2212 synthesis.

A similar increase in cinnamic acid hydroxylase (CAH)
occurs in response to ethylene treatments in pea seedlings
(26). This enzyme, which catalyzes the conversion of
cinnamic acid to p—coumaric acid, is increased when exposed
to 43 ul 1'1 ethylene. Its induction period is about 14
hours, with maximum activity occurring at 30 hours. CAH
inhibition requires more cycloheximide or actinomycin-D than
does PAL inhibition.

In genera14,jp1ant responses to ethylene are saturated
at concentrations of 10 ul 1"1 (l). Anthocyanin
biosynthesis in apple fruits is saturated at ltx>5 ul 1"1
ethylene (8). Therefore, the rate (Hf anthocyanin
biosynthesis in ripe apples, which contain 25 to 2500 ul 1'1
ethylene (4), is unaffected by additional ethylene

treatments (7, 19%. In unripe apples, ethylene increases

8

the rate of anthocyanin accumulation 3 to 4 fold and PAL
levels 3 to 6 fold (19). Final levels of anthocyanin are
similar to those observed in ripe apples (19). In fig,
ethephon similarly increases the rate of pigment formation
but does not affect the total amount accumulated (42).

Temperature An inverse relationship exists between

 

temperature and anthocyanin biosynthesis (l3,]£h.55, 57).
In ‘McIntosh' apples, rapid anthocyanin formation is
correlated to the onset of cool weather (13). Creasy (13)
suggests that the lower temperatures reduce sugar loss,
thereby increasing the rate of anthocyanin biosynthesis.
This hypothesis is suspect because: 1) his data do not
reflect large differences in fruit.sugar contents between
temperature treatments (13), and 2) fruit from heavily
cropped trees color and ripen later than fruit from lightly
cropped trees subjected to the same temperature regimes.
Under such circumstances, the stage of maturity may have had
a greater effect on anthocyanin formation than low
temperature (19).

High night temperatures may be more inhibitory to
anthocyanin biosynthesis than high day temperatures (57).
High temperature PAL inactivation, rather than low
temperature PAL induction is probably dominant in regulating
PAL levels (18). 'Pan (55) reports higher levels of the PAL-
inactivator in apples held at 18 C than at 6 C. Reduced
exposure of fruit to light leads to less induction of PAL;
darkness leads to PAL inactivation (18). High, dark

temperatures promote more PAL inactivation than low; dark

9
temperatures. This further supports the hypothesis that

anthocyanin biosynthesis is most inhibited due to high night
temperatures (18).

Cultivar Apple fruit coloration is dependent on a cultivar

 

and light interaction (29, 32, 41, 47). ‘McIntosh' apples
require 25 to 30% sky (32) or 50% full sunlight (29) to
develop acceptable color. Anthocyanin biosynthesis is
unaffected, however, inlfliller Sturdeespur ‘Delicious'tnr
95% shading from 55 days postbloom through harvest (47).
Another strain of ‘Delicious', Vance, has significantly
less anthocyanin biosynthesis when shaded 85% from 30 days
postbloom through harvest (41). Uncovering this fruit 20 to
30 days before harvest increases anthocyanin levels to those
levels present in fruit exposed to full sunlight throughout
the period (41). Similarly treated ‘McIntosh' have
significantly higher levels of anthocyanin than those
exposed to light continually (41). For both ‘McIntosh' and
Vance strain of ‘Deliciousfl,a.one day Unrabout 13 hours
sunlight) lag period is reported for anthocyanin formation
following uncovering. This lag period is consistent with
earlier findings (40, 54).

Growth Regulators Two types of growth regulators are used

 

 

to promote anthocyanin biosynthesis in apple and other
fruit. The first type delays fruit maturation, and thus
extends the growing season into cooler weather. The second
induces ethylene production or undergoes solvolytic
fragmentation to ethylene (22).

Daminozide (Alar) belongs to the first category. When

10
applied to apple trees 60 days before harvest, daminozide

delays the onset of the ethylene climacteric by 5 to 11 days
(15, 30, 33, 56). Fruit firmness and color are enhanced
(15, 23, 30), but fruit weight or size may be decreased
(34). Fruit drop is also reduced (15, 30).

Stop-drop sprays such as fenoprop (2,4,5-TP) or NAA may
induce ethylene production via auxin-induced ethylene
biosynthesis. Ethephon (2-chloroethylphosphonic acid) is an
ethylene-releasing plant growth regulator. In general,
ethylene-inducing and ethylene-releasing growth regulators
shorten the growing season by enhancing ripening because
they are applied 2 to 3 ‘weeks preharvest (10, 23, 35, 39,
52, 53, 56). They may consequently limit fruit size (10)
because the cell enlargement stage is shortened.
Anthocyanins are generally increased by ethylene-inducing
and ethylene—releasing compounds (15, 22), but because
ripening is also advanced, flesh firmness and shelf life
decrease (10, 15, 39). Ethephon causes fruit abscission,
therefore, an accompanied stop-drop spray is needed (15).

The ripening response to ethephon can be partially
counteracted by daminozide. Daminozide decreases the rate
of loss of flesh firmness and storage life caused by
ethephon treatments (15, 33, 34) and has an additive effect
on anthocyanin development (34L. Consequently, a preharvest
daminozide application is recommended whenever ethephon is
used.

Even when daminozide is used, response to ethephon is

often erratic (56). Solvolytic fragmentation of ethephon is

11

very temperature dependent because ethephon has an energy of
activation of about 30 kcal mol'1 (2, 38). At 25 C,
decomposition is slow, even in‘basiclsolutions (2L. It is
unlikely that the cell pH of apples is higher than pH 7.5
(45, 46). This below optimum pH, coupled with large
temperature fluctuations characteristic of late summer and
early fall, often results in ethephon—induced autocatalytic
ethylene production. Fruit ripening processes are initiated
concomitantly.

CGA 15281 (2-rfltloroethylmethyl-bis-phenylmethoxy-
silane) is, in contrast to ethephon, an unstable ethylene-
releasing plant growth regulator (51). It has an energy of
activation of only 10 kcal mol'1 (37) and is therefore much
less sensitive to temperature fluctuations. Since
solvolytic fragmentation of CGA 15281 is rapid in both
acidic and basic pH regimes (6), the cell vacuole may be a
favorable fragmentation site. Recent research with CGA
15281 confirms that solvolytic fragmentation occurs rapidly
in apple fruits; however, fruit ripening may also be
enhanced when CGA 15281 is used at higher concentrations

(11).

Objectives of Present Study
Previous research indicates that anthocyanin
biosynthesis in apple fruits can be regulated in the orchard
by light and ethylene treatments. The present study was
designed to determine: 1) how ethylene and light interact

in anthocyanin formation :hi apple fruits under field

12

conditions; and 2) if fruit coloration can be enhanced

using ethylene-releasing plant growth regulators without

stimulating fruit ripening. The results of these studies

are found in the following papers.

10.

ll.

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Engelsma, G. 1970. A comparative investigation of the
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Faust, M. 1965. Physiology of anthocyanin development
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Hort. Sci. 102 (4):491-494.

 

Grisebach, H. 1957. Zur Biogenese des Cyanidins. &
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Hyodo, H. 1971. Phenylalanine ammonia-lyase in
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Hyodo, H. and S. F. Yang. 1971. Ethylene-enhanced
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Johnson, T. and D. R. Dilley. 1968. The influence of
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Lakso, A.IL 1980. Correlations of fisheye photo-
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Liu, F. W. 1979. Interaction of daminozide, harvest-
ing date and ethylene in CA storage on ‘McIntosh' apple
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Looney, N.1L 1975. Control of ripening in ‘McIntosh'
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Massey, L.DL 1972. Ethephon: a postharvest aid to
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15

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108:573-574.

Olien, W. C. 1980. Ethephon-induced gummosis in sour
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function and influence of temperature» PhJL Thesis,
Michigan State Univ., East Lansing.

 

Olien, W. C. and M. J. Bukovac. 1978. The effect of
temperature on the rate of ethylene evolution from
ethephon and from ethephon-treated leaves of sour
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Pollard, J. E. 1974. Effects of SADH, ethephon and
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Proctor, J. T. A. and L. L. Creasy. 1971. Effect of
supplementary light on anthocyanin synthesis in
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526.

 

Proctor, J. T. A. and E. C. Lougheed. 1976. The
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Puech, A” A., C. A. Rebeiz and J. C. Crane. 1976.
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SECTION II.
EFFECT OF SHADING AND LOW ETHYLENE CONCENTRATIONS ON

ANTHOCYANIN BIOSYNTHESIS OF ‘PAULARED' APPLES

Abstract

Low concentrations of 2-chloroethylmethyl-bis-
(phenylmethoxy)-si1ane (CGA 15281) were applied two weeks
before estimated harvest maturity to ‘Paulared' trees.
Black shading cloth which eliminated 51%, 73% or 92% of full
sunlight was wrapped around spurs bearing individual fruits.
At 0, 5, 8 and 11 days after shading, fruit weight, size,
flesh firmness, anthocyanin content and internal ethylene
measurements were taken. CGA 15281 had no significant
effect, but shading decreased fruit size, weight and
anthocyanin, and increased flesh firmness relative to fully
exposed fruit. These differences were not attributed to
differences in fruit maturation as shading treatments had no
significant effect (n1 starch degradation run: internal
ethylene concentrations. The role of high temperature, low
light levels and ekhylene in anthocyanin biosynthesis and

fruit maturation is discussed.

High summer temperatures frequently suppress
anthocyanin formation 1J1 early apple cnfiltivars by
inactivating phenylalanine ammonia-lyase (PAL), the rate
limiting enzyme involved in anthocyanin biosynthesis (6).
The cultivar ‘Paulared', an important late summer apple
grown in Michigan, perhaps colors poorly for this reason.
Because of this, ethephon (2-chloroethylphosphonic acid) is
commonly used to enhance coloration, presumably via
ethylene-enhanced PAL synthesis. This compound often

promotes fruit ripening, however, as its degradation is
19

20
very temperature dependent (l, 11). Furthermore, some

growers apply it at rates above those recommended with
false hopes that ilzrmill substitute for good pruning
practices which enhance light penetration.

In the present work, the effects of ethylene and
shading on anthocyanin formation in the late summer cultivar
‘Paulared' were investigated. Because fluctuating
temperatures are common near harvest, we chose to use a
much less temperature dependent ethylene releasing compound,
CGA 15281 (2-chloroethylmethyl-bis-(phenylmethyoxy)-silane)

instead of ethephon.

Mater ia 1 s and Methods

 

Experimental design Twenty-four 8-year old ‘Paulared' apple

 

trees were selected at a commercial orchard in Greenville,
Michigan. ‘The trees were of uniform vigor, size and crop
load. Four concentrations of CGA 15281 were assigned at
random to six whole trees each. Each tree was further
divided into quadrants and four shading treatments were
assigned so that none would occur more than once per tree.
The experimental design was a split-split plot with chemical
concentration in the whole plot, shading level in the
subplot and sampling time in the sub-subplot.

Harvest date prediction Before chemical treatment, we

 

predicted the optimum harvest date of the fruit based on
long term CA storage recommendations (4). Ten uniform,
blemish-free apples were harvested at random from trees and

placed into 5.8 1 plastic containers. An envelope of

21

hydrated lime was placed into each to remove respiratory
C02, a competitive inhibitor of ethylene action. At daily
intervals, 1 ml gas samples were removed from each sealed
container through a rubber septum fitted into the lid.
These were analyzed using a Varian Aerograph Series 1700 gas
chromatograph (Varian Instruments, Walnut Creek CA) equipped
with a 1 m activated alumina column and flame ionization
detector. Upon induction oftfluaethylene climacteric, or
when 0.5 ul 1'1 ethylene had accumulated within the
container, the number of hours from sealing were recorded
and multiplied by a constant of 0.125 days per hour (3).
The product is the number of days from sampling to the
estimated optimum harvest date in Michigan for long-term CA
storage» This method was used in additionito the standard
prediction methods based on temperature and days from full
bloom.

Chemical treatment Two weeks before the estimated harvest

 

date (20 August 1984), the following concentrations of CGA
15281 were applied to the respective whole trees as
designated: 0 mM, 0.125 mM, 0.250 mM and 0.500 mM CGA
15281. All tank mixtures (including 0 mM control) contained
X-77 (alkyl aryl polyethoxy ethanol and free fatty acids,
Chevron Chem. Corp., San Francisco CA) and Fruitone-T
(2,4,5-TP, Union Carbide Agricultural Products Co., Inc.,
Ambler PA). Both were added at the recommended rate of 0.625
ml 1'1.

Shading treatment Black polypropylene fabric (Chicopee

 

Manufacturing Co., Chicopee GA) which eliminates 51%, 73%

22
or 92% of full sunlight was cut into 25 x 36 cm pieces. One

day after chemical application, shading cloth was wrapped
and stapled around spurs of individual fruits within the
proper quadrant. The remaining uncovered quadrant of each
tree was the 0% shading (or full sunlight) treatment.

Quality and maturity evaluations A minimum of 10 fruits per

 

treatment and replication were harvested at 0,5L.8 and 11
days after fruit shading to assess fruit quality and
maturity. In addition to fruit weight and diameter
measurements, a visual estimation of percent red surface
coloration was made to the nearest 10%. Skin discs (1.4 cm
diameter) were taken from the most highly colored cheek of
each fruit and total anthocyanins were extracted in 10 ml of
10% oxalic acid at 5 C. Upon extraction, total anthocyanins
were measured at 530 nm spectrophotometrically; Due to some
shade cloth induced fruit scalding, only six fruits per
treatment and replication were used for these evaluations
after the first sampling time. Flesh firmness was
determined using an Effigi penetrometer fitted withaalull
cm tip. Using the starch-iodine staining technique outlined
for ‘McIntoshfl apples, a starch index was assigned to each
fruit (13). Five fruits per treatment and replication were
used for these measurements at the first sampling time;
three fruits per treatment and replication were used at the
later sampling times. Internal fruit ethylene samples were
taken with a 1 ml syringe from the cavities of fruit at each
sampling time. Because the number of fruit sampled varied

between sampling times, this measurement was used to

23‘

determine differences in maturity between chemical and
shading treatments within sampling time only.

Statistical analysis All maturity and quality data were

 

subjected to an analysis of variance. Treatment effects
were determined using least significant difference (lsd)

values at the 5% level.

Results and Discussion

 

Chemical treatment Low concentrations (75 ppm and less) of

 

 

ethephon have been reported to stimulate coloration and
advance the harvest of ‘McIntosh' apples without sacrificing
fruit condition or storage life (2, 7, 14). Since
degradation of this compound to ethylene is very temperature
dependent, results are often unpredictable under field
conditions. CGA 15281, another ethylene releasing compound,
is an unstable silicon compound, having a much lower energy
of activation in contrast to ethephon (10).

The CGA 15281 concentrations used in this experiment
were approximately equimolar to ethephon concentrations used
with success on ‘McIntosh' earlier (2, 7, 14). In this
study, no significant differences in fruit coloration or
maturity were attributed to concentrations of 0.5 mM CGA
15281 or less when applied to ‘Paulared' apples 11 to 14
days before the optimum harvest date. Perhaps, the
‘Paulared' cultivar is less sensitive to ethylene than
‘McIntosh' or field temperatures were significantly higher
during the course of our experiment, leading to a net PAL

inactivation.

24
Shading treatment Zyll shading treatments affected fruit

 

size, weight, color and firmness. Fruit exposed to full
sunlight continued to grow atainearly linear rate during
the 11 days observed (Figure l). Shaded fruit ceased growth
during the first five days, but appeared to adapt and
increase in weight thereafter at a rate similar to the fully
exposed fruit. These results support the findings of others
(8, 9, 12,16) that shading reduces fruit size. However,
nonsignificant differences in fruit weights of several
artificially shaded cultivars have also been reported (15).

Firmness increased or remained the same during the
first five days, depending onifluashade treatment (Figure
2). This lack of softening has been reported elsewhere (8,
l6) and was attributed to a shade-induced cessation of fruit
growth and delayed maturation (8). Fully exposed fruit
continued to grow at a linear rate and did not differ
significantly from shaded fruit in ethylene concentration or
starch index. Firmness decreased as fruit weight increased
and shading affected both to about the same extent. Flesh
softening during growth is largely due1x>the increase in
intercellular airspace.

Anthocyanin content, expressed as absorbance at 530 nm
decreased significantly during the first five days in.all
treatments except the non-shaded and 51% shaded treatments
(Figure 3). Significant decreases, which probably reflected
anthocyanin turnover, occurred during the last 3 days in
fruit shaded 73% and 92%. Similar trends are seen in Figure

4 where percent red surface coloration was estimated

25

 

    

 

 

 

 

 

 

.—

l T l I l

1 i S 4 5 6 7 8
DAYS AFTER SHADING

150i)
H control
1 e—o 51% SHADE «
A ra—a 73% SHADE
E 140-0" H 927. SHADE 4
8 1
3 130 OH
g .
§ 120.0- —
s . *
Bf 110.0- 1
1 ~
01
0

Figure 1. Fruit weight
(n=l44 except at day 0 where n=960);
comparing within same sampling time,
shade level.

and

I
11

0, 5, 8 and 11 days after shading
lsd .05 =

6.80 for
5.87 within same

26

 

 

 

800

q 1
r\ /’_‘
m ‘ .
C
O ‘ _ ..
44 -
3 _ .
93 75.04 1
\./

‘1 d
m C
E . W «
2f ‘ .
2 .4 1
a:
l: 700- 1
I: ‘ .
B ‘ H 92% shade .
L—LJ 4 H 737: shade .

‘ke—o 51% Shade .

H control

 

 

O l l l l I T l l l I

012 3 4 5 6 7 8 91‘011
DAYS AFFER SHADING

Figure 2. Flesh firmness of ‘Paulared' apples 0, 5, 8
and 11 days after shading (n=72 except at day 0 where
n=480); lsd .05 = 2.00 for comparing within the same

sampling time, and 1.88 within same shade level.

within same

27

 

 

 

 

 

shade level.

(1900
'i H 4
o.SooJ 1
A
E « .
C
0.7004 -
O
B .. _.
V 0.600“ 3 -(
LIJ 4 e ..
(Z) 2
< 0.500“ ' -I
a) . g .
m -
O 0.400- - a -
(f)
g ‘ a—a 92% shade ‘
0.300— B—B 73% shade “
\e—o 51% shade
OJH control
I I I I I I I T I I I
O 1 2 3 4 5 6 7 8 9 10 11
DAYS AFTER SHADING
Figure 3. Absorbance at 530 nm of anthocyanin extracted
from ‘Paulared' apples 0, 5, 8 and 11 days after
shading (n=l44 except at day 0 where n=480); lsd .05==
.079 for comparing within same sampling time, and .076

 

28

 

control
51% shade 4
73% shade
92% shade

IEII

 

PERCENT RED COLOR

 

 

 

—

I I I r I r’ I I I
1 2 3 4 5 6 7 8 9 10 11

DAYS AFTER SHADING

O
C)--ll-—//l

Figure 4. Visual estimation of percent surface coloration
of ‘Paulared' apples 0, 5, 8 and 11 days after shading
(n=l44 except at day 0 where n=480); lsd .05 = 4.4 for
comparing within same sampling time, and 4.3 within same
shade level.

29

visually at each sampling time.

Fruit maturation was unaffected by shading as no
significant differences in starch indexes or internal
ethylene concentrations were found at any sampling date.
However, it was evident that the shading material not only
reduced light penetration, but also increased temperatures
surrounding the fruit as some fruit had to be discarded due
to scalding. This is important since decreased light
reduces PAL induction (5) and high temperatures inactivate
PAL (17). High temperatures, combined with reduced light or
darkness, are even more inhibitory as dg 2222 PAL synthesis
is reduced or inhibited and the amount of PAL-inactivator

is increased concomitantly (5).

Literature Cited

 

l” Biddle, E., D. G. S. Kerfoot, Y. H. Kho, and K. E.
Russell. 1976. Kinetic studies of the thermal
decomposition of 2-chloroethylphosphonic acid in
aqueous solution. Plant Physiol. 58:700-702.

 

2. Blanpied, G. D., C. G. Forshey, W. C. Styles, D. W.
Greene, W. J. Lord and W. J. Bramlage. 1975. Use
of ethephon to stimulate'red color without hastening
ripening of ‘McIntosh' apples. g; Amer. Soc. Hort.

 

Sci. 100(4):379-381.

3. Dilley, C. L. and D. R. Dilley. 1985. New technology
for analyzing ethylene and determining the onset of the
ethylene climacteriCIOf apples. pp.353-362. In: S.
M. Blankenship (Edd. Proc. 4th National Controlled
Atmosphere Research Conference. North Carolina State
University. Horticultural
Report No. 126.

4- Dilley, D. R. 1980. Assessing fruit maturity and
ripening and techniqueslx>delay ripening in storage.
110th Ann. Rpt. Mich. State Hort. Soc. 132-146.

 

10.

11.

12.

l3.

14.

15.

16.

3O

Faragher, J. D. 1983. Temperature regulation of
anthocyanin accumulation in apple skin. J_. Exp. Bot.
34(147):1291-1298.

 

Faragher, J.[L and D.£L Chalmers. 1977. Regulation
of anthocyanin synthesis in apple skin. III.
Involvement of phenylalanine ammonia-lyase. Aust. J;
Plant Physiol. 4:133-141.

 

Greene, D. W., W. J. Lord, W. J. Bramlage and F. W.
Southwick. 1974. Effects of' low ethephon
concentrations on quality of ‘McIntosh' apples. J;
Amer. Soc. Hort. Sci. 99(3):239-242.

 

Heinicke, D. R. 1966. Characteristics of McIntosh and
Red Delicious apples as influenced by exposure to
sunlight during the growing season. Proc. Amer. Soc.
Hort.Sci.89:l0-13.

 

 

Jackson, J. E. and J. W. Palmer. 1977. Effects of
shade on the growth and cropping of apple trees. II.
Effects on components of yield. & Hort. Sci. 52:253-
266.

 

Olien, W.CL 1980. Ethephon-induced gummosis in sour
cherry (Prunus cerasus): Effect of gum on xylem
function and influence of temperature. Ph. D. Thesis,
Michigan State Univ., East Lansing.

 

Olien, W. C. and M. J. Bukovac. 1978. The effect of
temperature on rate of ethylene evolution from ethephon
and from ethephon-treated leaves of sour cherry. J;
Amer. Soc. Hort. Sci. 103:199-202.

 

Perring, M. and H. Clijsters. 1974. The chemical
composition and storage characteristics of apples grown
in black cloth bags. gual. Plant. 23(4):379-393.

 

Phillips, W. R. and P. A. Poapst. 1952. Storage of
apples. Can. Dapt. Agr. Bul. 776.

 

Pollard, J. E. 1974. Effects of SADH, ethephon and
2,4,5-TP on color and storage quality of ‘McIntosh'
apples. J_._ Amer. Soc. Hort. Sci. 99(4):341-343.

 

Proctor, J. T. A. and E. C. Lougheed. 1976. The
effect of covering apples during development. HortSci.
11(2):108-109.

Robinson, T. L., E. J. Seeley and B. H. Barritt.
1983. Effect of light environment and spur age on
‘Delicious' apple fruit size and quality. g; Amer.
Soc. Hort. Sci. 108(5):855-861.

 

17.

Tan, S. C.

31

1979. Relationships and interactions

between PAL, PAL-inactivating system and anthocyanin

in apples.
586.

J; Amer. Soc. Hort. Sci. l04(5):581-

SECTION III.
ANTHOCYANIN BIOSYNTHESIS AND MATURITY OF ‘MCINTOSH'

APPLES AS INFLUENCED BY ETHYLENE-RELEASING COMPOUNDS

Abstract

Solvolytic fragmentation of 2-chloropropylphosphonic
acid, 2-chloroethylphosphonic acid (ethephon), «and 2-
chloroethylmethyl-bis-(phenylmethoxy)-silane (CGA 15281) was
studied. Based on laboratory results, ethephon and CGA 15281
were applied two or three weeks before the onset of
autogenous ethylene production in ‘McIntosh' apple.
Solvolytic fragmentation of the compounds and autocatalytic
ethylene production, anthocyanin biosynthesis, and flesh
firmness changes of the fruit were monitored. 2.0 mM (290
ppm) of ethephon applied at either date degraded at a slow,
constant rate resulting iJIIa significant increase in
anthocyanin biosynthesis within 6 days. However,
autocatalytic ethylene production and flesh softening
accompanied this increase. CGA 15281 generated ethylene
within 3 hours of application. Ethylene levels of fruit
treated with 1.0 mM CGA 15281 three weeks before harvest
returned to low levels within 24 to 48 hours of application.
Anthocyanin increased significantly within 6 days; this was
not accompanied by autocatalytic ethylene production or
accelerated flesh softening. Results indicate that
anthocyanin biosynthesis can be enhanced in apples using

ethylene-releasing compounds without inducing ripening.

The ‘McIntosh' apple is one of the most important
cultivars grown in the United States, ranking third in total
production. Due to poor coloration, however, a significant

percentage of the fruit harvested each year fail to meet the
33

34
U. S. Extra Fancy grade.

Cultivar, light, temperature and ethylene influence
anthocyanin biosynthesis in apple skin (2, 3, 6, 9, 17). A
light requirement must first be met, afterwhich anthocyanin
biosynthesis is apparently limited by phenylalanine ammonia—
1yase (PAL) (8). Ethylene enhances PAL activity in several
tissues, including preclimacteric apples (7). High
temperatures appear to inactivate PAL Min; however, the
effect of temperature depends largely upon the stage of
fruit maturity (6).

2-chloroethylphosphonic acid (ethephon), an ethylene-
generating compound, is used commercially to enhance
anthocyanin development in McIntosh and other early apple
cultivars. Response to this compound is often erratic
however, due to its relatively high energy of activation.
At 25 C, decomposition is slow, even in basic solutions (1).
Because of this, ethephon applied to trees with mature fruit
often induces autocatalytic ethylene production. Ripening
processes are initiated concomitantly, resulting in rapid
cell wall breakdown and fruit softening.

Since development of the anthocyanin biosynthetic
capacity requires only a brief exposure to ethylene or an
olefin, an ethephon-like compound having a lower energy of
activation is desirable. Anthocyanin biosynthesis could
then be enhanced without sacrificing postharvest integrity.
In a preliminary field study conducted in 1983, two unstable
compounds, 2-chloropropylphosphonic acid and 2-chloroethyl-

methyl-bis-(phenylmethoxy)-silane (CGA 15281) were compared

35
to ethephon applied at similar rates. Degradation of the

former (propylene-releasing) compound probably occurred
prior to absorption as it did not affect anthocyanin
biosynthesis” CGA 15281, however, stimulated anthocyanin
development more quickly than ethephon and without any
apparent stimulation of ripening.

In the present study, we followed the time course of
ethylene generation from ethephon, 2-chloropropy1phosphonic
acid, and CGA 15281 under controlled pH and temperature
regimes. A similar field study with McIntosh was conducted
using only ethephon and CGA 15281. Autocatalytic ethylene
production and postharvest changes of the fruit were
monitored. We wished to determine if anthocyanin
biosynthesis could be enhanced using ethylene-releasing

compounds without stimulating fruit ripening.

Materials and Methods

Sglyglytic fragmentation 2f ethylene- and propylene-

 

 

 

generating compounds Twenty airtight polypropylene

 

syringes (60 ml capacity) were used as vessels. Each was
filled with 50 m1 of ethylene-free air and immediately
sealed.with tightly fitting serum caps.TmnIml of citrate-
phosphate buffer of pH 3.0, 4.0, 5.0, 6.0, or 7.0 was
injected into quadruplicate vessels. The volume and pressure
changes due to the addition of buffer were compensated for
by adjusting the plunger to the 60 ml mark.

Ethephon, 2-chloropropy1phosphonic acid, or CGA-15281

were injected via ndcrosyringe from .1% stock solutions to

36
one pH series each. The resulting concentration in 10 mls of

buffer was 2.23 mMg Upon complete solvolytic fragmentation,
5 ul of ethylene or propylene would be released into the
headspace of the vessels, disregarding the amount of gas
dissolved in the buffer. The fourth pH series was used as a
control.

The vessels were held at 20 C and gas samples were
taken in duplicate with lrnl syringes at 1.5, 3, 6, 12Iand
24 hours. The headspace of each vessel was reduced by 2 ml
to compensate for volume and pressure changes each time.
Gas samples were analyzed using a Varian Aerograph Series
1700 gas chromatograph (Varian Instruments, Walnut Creek CA)
equipped with a 1 m activated alumina column and flame
ionization detector.

The experiment was a split-plot design with chemical
and pH in the whole plot and time of sampling in the
subplots. The experiment was replicated three times.

Field study Ten mature McIntosh trees bearing similar crop

 

loads were selected an: the Michigan State University
Horticultural Research Center in East Lansing, Michigan.
Each tree was divided into fifths and chemical treatments
were assigned so that none would occur more than once per
tree or location within the tree. The general experimental
design was a split-split plot with treatment time in the
whole plot, chemical treatment in the subplot, and sampling
time in the sub-subplots.

Harvest date prediction Before chemical treatment, we

 

predicted the optimum harvest date of the fruit based on

37

long term CA storage recommendations(5). Ten uniform,
blemish-free apples were harvested per tree at elbow depth
and placed into 5.8 1 plastic containers. An envelope of
hydrated lime was placed into each to absorb respiratory
C02, a competitive inhibitor of ethylene action. At daily
intervals, 1 ml gas samples were removed from each sealed
container through a rubber septum fitted into the lid.
These were analyzed by gas chromatography. Upon induction
of the ethylene climacteric, or when 0.5 ul 1‘1 ethylene had
accumulated within the container, the number of hours from
sealing were recorded and multiplied by a constant of 0.125
days per hour (4). The product is the number of days from
sampling to the optimum harvest date in Michigan for long-
term CA storage. This method was used in addition to the
standard prediction methods based on temperature and days
from full bloom.

Chemical treatment Three weeks before the estimated

 

 

harvest date (19 September 1984), the following treatments
were applied tn) the respective tree sections: 2.0 mM
ethephon, 2.0 mM CGA 15281, 1.0 mM CGA 15281, 0.5 mM CGA
15281 and control. All tank mixtures (including control)
contained both X-77 (alkyl aryl polyethoxy ethanol and free
fatty acids, Chevron Chem. Corp., San Francisco CA) and
Fruitone-T (2,4,5-TP); both were added at the recommended
0.625 m1 1'1 rate. The same protocol was followed one week
later when the second main plot was treated. Both chemical

applications were completed between 8 and 9 am.

38
Solvolytic fragmentation 9f CGA 15281 and ethephon lg apples

 

 

At 0, 3, 6, 12, 24, 48, and 96 hours after application,
internal ethylene samples were taken with a 1 ml syringe
from the cavities of five fruits per replication and
treatment. Solvolytic fragmentathma of the ethylene-
releasing chemicals was monitored by gas chromatography.
Weather data recorded at the Research Center were used to
interpret chemical absorption and degradation.

Qualitj and maturity evaluations A minimum of 15 fruits

 

per treatment and replication were harvested at 0, 3, 6, 9,
12 and 15 days after the first application and through 12
days following the second. Five fruits from each sample
were used to assess fruit quality and maturity at each time.
In addition to fruit weight and diameter measurements, a
visual estimation of the percent red surface coloration was
made to the nearest 10%. Flesh firmness was determined
using an Effigi penetrometer fitted with a 1.11 cm tip.
Using the starch-iodine staining technique outlined for
McIntosh apples (l3),arstarch index was assigned to each
sample. Skin discs (LA cm diameter) were taken from the
most highly colored cheek of each fruit and total
anthocyanins were extracted in 10 ml of 10% oxalic acid at 5
C. Upon extraction, total anthocyanins were measured at 530
nm spectrophotometrically. The remaining ten or more fruit
were stored in paper bags at approximately 20 C. Flesh
firmness measurements were repeated in the same way at one

and two weeks postharvest and the relative shelf life of

fruit from each treatment was assessed.

39

All maturity and quality data were subjected to an
analysis of variance and trend analysis. Treatment effects
were determined using least significant difference (lsd)
values at the 5% level. Rates of anthocyanin biosynthesis
and fruit maturation were examined using linear regression;
t values were computed and used to compare differences

between treatment slopes.

Results and Discussion

Igglyglytic fragmentation 3f ethylene- and propylene-

 

 

 

generating compounds_ig‘vitro Solvolytic fragmentation was

 

highly dependent on solution pH for all compounds studied.
2-chloropropy1phosphonic acid buffered at pH 5.0,(L0 and
7.0 degraded completely within 1.5 hours (see Figure 1A).
These results explain, at least in part, our preliminary
field results which showed no stimulation of anthocyanin
biosynthesis by this compoundaIComplete degradation probably
occurred before chemical absorption by the fruit, or
possibly in the mixing tank. Because this compound appeared
to be too unstable, it was not included in the 1984 field
study.

CGA 15281 released ethylene more readily as acidity
increased from pH 7.0 to 3JL In contrast, ethephon was
stable at pH 3.0, 4.0 and 5.0 (see Figures 18 and 1C). At
pH 7.0, the amount of ethylene generated by both compounds
was similar at each sampling time. However, neither

compound underwent more than 30% solvolytic fragmentation at

40

Figure 1. Solvolytic fragmentation of .A) 2-
chloropropylphosphonic acid, B) CGA 15281, and C)
ethephon in citrate-phosphate buffers adjusted to pH 3, 4,
5, 6 and'L. Incubation was at 20 (2(n=6).

Figure 1.

PERCENT FRAGMENTATION TO GAS

PERCENT FRAGMENTATION TO GAS

'1 \
11.1

PERCENT FRAGMENTATION TO GAS

 

41

 

 

 

 

 

moi—W"
90‘ k \. .-
ao- ‘l
‘ -l
70: 2-CHLOROPROPYLPHOSPHONIC ACID 1
pH .0 1
pH .0 -
pH .0 ‘1

pH .0
6 pH .0 ‘
v I f I 1 T v T fl I ' I 7 T j r
o 3 a 9 12 1s 18 21 24

 

HOURS AT 20 C

 

O) \J m (D O
O O O O D
1 L l 1 14 J L l

A

U"
C
l

A

A
O
1

L

E

CGA 15281

I

//\+

 

 

 

*1 r v r v j Y T 1

S 9 12 15 18 21 23
HOURSAT20C

 

100‘

pH 3.0 l
pH 4.0
pH 5.0
pfliiO
pH‘ZO

O

AJ—LillLi

ETHEPHON

IIIII

 

J_lnlnlAlAlAl

 

 

 

 

3
HOURS AT 20 c

42
pH 7.0 when held at 20 C.

Solvolytic fragmentation pf CGA 15281 and ethephon ip apples

 

 

Several pH regimes which would affect the'rate of solvolytic
fragmentation would be encountered as these compounds are
absorbed into the cell. In maize, the cytoplasmic pH is in
the range of 7.0 to 7.5, and the pH of the vacuole about pH
5.0 to 5.5 (14,15). The cell wall probably has an
intermediate pH. Due to the high concentration of malic
acid found in apple fruits, however, we would expect the pH
of these cell compartments to be more acidic. Therefore,
the expected site of ethephon fragmentation would be
largely cytoplasmic while fragmentation of CGA 15281 would
be more favorable in the cell wall and vacuoleu The rate of
solvolytic fragmentation in the cytoplasm would be
approximately equal for both compounds. Overall, the rate
of ethylene release from CGA 15281 would probably be more
rapid than that from ethephon in apple fruit tissue.
Internal ethylene concentrations of treated fruit are
shown in Figures 2A and 28. Solvolytic fragmentation of CGA
15281 to ethylene peaked about 6 hours after application on
August 29, or three weeks before the predicted optimum
harvest date. Internal ethylene concentrations were
proportional to the concentration ofCXH115281 applied on
this date. With the exception of the 2.0 mM treatment,
internal ethylene concentrations returned to low levels
within 24 to 48 hours of application. Ethephon degradation
was slow, having no obvious "peak" time of ethylene release.

Apparentlyy the internal ethylene concentrations of these

43

Figure 2. Solvolytic fragmentation to ethylene of CGA 15281
and ethephon applied to mature ‘McIntosh' trees on A)
29 August 1984 or B) 6 September 1984. Solvolytic

fragmentation was measured as internal fruit ethylene by
gas chromatography (n=25).

INTERNAL ETHYLENE (HI/I)

INTERNAL ETHYLENE (pl/l)

44

 

3.500
4 8/29 APPLICATION @303 2A.

 

 

 

 

 

 

0.000 VIV'IYT:T—I'TIITV____——————
0 12 24 36 48 60 72 84 96

HOURS AFTER APPLICATION

 

9/6 APPLICATION {"6" (""9‘ 28

 

l

Lg

L

d

1

d

d

 

 

 

 

 

'[TrYITTrIVT4

o 12 24 36 48” so 72 84 6
HOURS AFTER APPLICATION

 

Figure 2.

11311

IZEII

2.0 mM ETH
2.0 mM CGA
1.0 mM CGA
0.5 mM CGA
control

2.0 mM ETH
2.0 mM CGA
1.0 mM CGA
0.5 mM CGA
control

45
fruit, as well as those treated with 2.0 mM CGA, remained

above the ambient level characteristic of preclimacteric
fruit; within 2 to 4 days of application, autocatalytic
ethylene production was induced by both treatments.
Autocatalytic ethylene production did not occur until 12 to
15 days following treatment with the lower CGA
concentrations (data not shown).

When applied to trees on 6 September, or two weeks
before the predicted harvest date, we saw no well defined
ethylene peaks due to chemical degradation (see Figure ZB).
This difference in response between application dates is
most likely due to the much cooler temperatures, i.e. a
maximum of 32.2 C on 29 August vs. 18.31C on September 6.
Lower temperatures would account for. slower rates in
chemical degradation (1,11,12), and chemical absorption
(10). With temperatures rising slightly during the
following days, internal ethylene concentrations of fruit
treated with 2.0 mM CGA 15281 or ethephon also rose. From
the data, we cannot postulate whether the increase in
internal ethylene observed at 48 hours after application was
due to a delay of solvolytic fragmentation or if this was
autocatalytic ethylene production. At 96 hours, however, it
was apparent that the fruit had entered the ethylene
climacteric.

Quality and maturity gyglpggippg No significant

 

 

differences in fruit weight or diameter were attributed to
chemical treatment. Anthocyanin content, expressed as

percent surface coloration and total anthocyanins extracted,

46

increased over time but also depended on chemical treatment
(see Figures 3A, 3B, 4A, and 4B). Regardless of the date
applied, anthocyanin increased significantly in fruits
treated with 1.0 mM or 2.0 mM CGA 15281, and 2.0 mM
ethephon. Significant increases occurred within 6 days of
the 29 August application. Fruit treated one week later
responded similarly, except that the 1.0 mM CGA 15281
treatment did not promote a significant increase in
anthocyanin until 9 days after application. Anthocyanin
increases over time were generally nonsignificant in control
fruit and fruit treated with 0.5 mM CGA 15281. Since highly
significant linear trends characterized anthocyanin
biosynthesis over time, we used linear regression to define
the rate increases. At both application dates, a t test
indicated that anthocyanin biosynthesis occurred at
significantly greater rates in fruit treated with 1.0 mM CGA
15281 than control or 0.5 mM treatments. Both the 2.0 mM
ethephon and CGA 15281 treatments promoted higher rates of
anthocyanin biosynthesis than the 1.0 mM CGA 15281
treatment, however.

Flesh firmness also depended on an interaction of
concentration and sampling time (see Figures 5A and SB).
Fruit treated with either 2.0 mM ethephon or 2.0 mM CGA
15281 three weeks prior to optimum harvest were
significantly softer than control fruit after 9 and 12 days,
respectively. Fruit treated with the same rates on
September 6 were significantly softer within only 6 days.

Twelve days after this application, fruit treated with 1.0

47

Figure 3. Percent red coloration of ‘McIntosh' apples
treated with CGA 15281 or ethephon on A) 29 August 1984

orB) 6 September 1984. Red surface coloration was
estimated visually to the nearest 10% (n=25); lsd .05
within sampling date = 12.939 (A) and 13.450 (B), and

within concentration = 11.260 (A) and 13.133 (B).

48

 

 

 

 

 

 

 

 

 

 

100 7
H control 3A
~ o—e 0.5 mM CGA 8/29 APPLICATION 1
B—El 1.0 mM CGA
o: 80* H 2.0 mM CGA s
9 H 2.0 mM ETH
O 'I
Q
60- .1
B ,.
LI: v: a 1
I—
Z ..
0: Th -——-;/
LIJ
0- _
O I I I I I
0 3 6 9 12 15
DAYS AFTER CHEMICAL APPLICATION
100 38
H control
H (,5 mM CGA 9/6 APPLICATION ,
H 1.0 mM CGA
or 80* H 2.0 mM CGA -\ a
53 k—t ZADrnM ETH T“ 4
o 1
Q) 60 3
D :-
LIJ :
0f. 3 ..
g 4.- , = . _
g 4 / V .
LLI
0- 2o -
O T T I I T
0 3 6 9 12 15

DAYS AFTER CHEMICAL APPLICATION

Figure 3.

49

Figure 4. Absorbance of anthocyanin extracted from
‘McIntosh' apples treated with CGA 15281 or ethephon on A)
29 August 1984 or B) 6 September 1984 (n=25); lsd .05
within sampling date = .126 (A) and .145 (B), and within
concentration =.119 (A) and .136 HH.

ABSORBANCE (530 nm)

ABSORBANCE (530 nm)

Figure

50

 

 

 

 

 

 

 

 

 

1.200
H control 4A
‘ G—O 0.5 mM CGA ‘
10004 H 1.0 mM CGA a
H 2.0 mM CGA
‘ t—A ZIJrnM EH4
0.800— ~
4 /
0.60% 2 a
0.4004 ,- a ' ..
J / T a
CLZOO “———'%"’ 4
fl 8/29 APPLICATION
0000 I I I I T
O 3 6 9 12 15
DAYS AFTER CHEMICAL APPLICATION
1.200
H control 48
0—0 0.5 mM CGA T
1.00m a—a 1.0 mM CGA d
A—A 2.0 mM CGA
I H 2.0 mM ETH 4..
O.800~ -
0.600- / . -
0.40m / <-‘ ~
0.2004 ~
. 9/6 APPLICATION
0000 r I I I I
0 3 6 9 12 15

DAYS AFTER CHEMICAL APPLICATION

51

Figure 5. Flesh firmness of ‘McIntosh' apples at each
sampling date treated with CGA 15281 or ethephon on A) 29
August 1984 or B) 6 September 1984 (n=25); lsd .65
within sampling date = 3.893 UH and 3.069 U”, and within
concentration = 3.597 (A) and 3.169 (B).

52

90.0

 

8/29 APPLICATION

   
  

80.0—

70.04

60.0—4

 
  

2.0 mM ETH
2.0 mM CGA
1.0 mM CGA
0.5 mM CGA
control

50.0 I I T I I
0 3 6 9 12 15
DAYS AFTER CHEMICAL APPLICATION

IIEZI

 

 

 

FLESH FIRMNESS AT HARVEST (newtons)

90

 

58
. 9/6 APPLICATION

80~ d

;§j
70 - \;’ ..

60

l

H 2.0 mM ETH . -
H 2.0 mM CGA

H 1.0 mM CGA

o—e 0.5 mM CGA

H control

50 I I I T I
0 3 6 9 12 15

DAYS AFTER CHEMICAL APPLICATION

 

 

 

FLESH FIRMNESS AT HARVEST (newtons)

Figure 5.

53
mM CGA 15281 were significantly softer than the controls.

Linear trends best characterized the responseeto chemical
treatment over time. Comparing slopes of the regression
lines, we found no significant differences in softening
rates of fruit treated with 1.0 mM CGA 15281 or control.
Fruit treated with 2.0 mM ethephon on August 29 and
6 September, respectively, softened at significantly greater
rates than fruit treated with 1.0 mM CGA 15281, however.

Figures 6A and GB illustrate the shelf life of fruit
harvested 9 days after chemical treatment. With fruit
treated on 29 August, no interaction was present between
chemical treatment and sampling time, but both main effects
were highly significant. The rates of softening were thus
nonsignificant ix} fruit treated (n: this date. Fruit
treated with either 2.6 mM CGA 15281 or ethephon on
September 6 were also initially softer than fruit of other
treatments 9 days after application, however, after 2 weeks
at:20 C, firmness values were not significantly different
between treatments. Rates of softening were significantly
greater in fruit from control, 0.5 mM and 1.0 mM CGA 15281
treatments.

Starch conversion to sugar also increased significantly
in fruit treated with 2 mM CGA 15281 and ethephon (see
Figures 7A and 7B). Starch conversion was linear and
occurred at rates dependent on an interaction of chemical
treatment and time. Significant treatment differences were
observed within 9 days of the 29 August application and 6

days of the 6 September application.

54

Figure 6. Flesh firmness at G, l, and 2 weeks of ‘McIntosh'
apples harvested 9 days after treatment on A) 29 August
l984or B) 6 September 1984. Fruit was stored at 20 C
(n=25); lsd .65 within sampling week = 3.737 (A) and 2.559
(B), and within concentration = 3.682 (A) and 2.199 (B).

55

 

 

 

 

 

 

 

 

 

80.0 F ' ' ' ~
A . 8/29 APPLICATION
Z T\ ‘
V70.0 ~ -
U)

m I .
L213 60.0 . _
LI 50:0 ~ 4
I ’ ~——~ CONTROL I
8 40.0 A o———o 0.5 mM CGA -
__. a——a 1.0 mM CGA
LI. V A————A 2.0 mM CGA ‘
O OIL *“T‘ 2'0 "‘M ET“ . 6&-
IhHTbAL '7 ENVYSL 14»IDAHTS
DAYS AT 20 C

80.0 F‘ F 1 "
A L 9/6 APPLICATION
23
V70.0 ~ A
(f)

m * \ I
§6QOP ~
5 I ‘
L 50.0 ” T\\ T
I I ~———- CONTROL ‘
LU)“ 40-OI' o—a 0.5 mM CGA 4
_J a———a 1.0 mM CGA
u. ‘ .————. 2.0 mM CGA ‘
0 0‘? .——. 2.0 mM ETH l 681 .
thTLAL. 7'IDANTS 1‘1 DMVHS

Figure 6.

DAYS AT 20 C

56

Figure 7. Starch index of ‘McIntoSh'apples treatedeith
CGA 15281 or ethephon on A) 29 August 1984 or B) 6
September 1984 (n=25); lsd .05 within sampling date = .853
(A) and .846 (B), and within concentration = .763 (A) and

.796 (B).

57

 

 

 

 

 

 
  
   

 

 

 

91)
.I H control 7A.
8.0— 9—0 0.5 mM CGA . ._
.I a—a 1.0 mM CGA ..
7.0. H 2.0 mM CGA ..
H 2.0 mM ETH .I
33 6.0~ A
o . "' .
Z 5.0- _
I J o .I
O — . .4
SE 413‘ :’_ .
5 :50— / —
2 OT / “
-I /—-
1.0
. 8/29 APPLICATION
00 I I I I I
0 3 6 9 12 15
DAYS AFTER CHEMICAL APPLICATION
91)
i H control 78 A
8.0- o—o 0.5 mM CGA -
.I B—EJ 1.0 mM CGA .
7.04 H 2.0 mM CGA _
H 2.0 mM ETH
5,3 6.0- -
Q .
Z 5.0a —
I . .
g 4 O"I 'I
S .
U) 3.0~ -I
21) -
* I
1.0- d
I 9/6 APPLICATION
0-0 I T I I I
0 3 6 9 12 15

 

DAYS AFTER CHEMICAL APPLICATION

Figure 7.

58
These observations were consistent with others that

climacteric fruits become more sensitive, and consequently,
more responsive to ethylene as they mature. This creates a
problem, therefore, when ethylene-releasing compounds such
as ethephon are applied near harvest to enhance fruit
coloration. Temperatures in the orchard at this time (late
summer and early fall) fluctuate greatly. Having an energy
of activation of about 30 kcal mol"l, the solvolytic
fragmentation of ethephon is highly dependent on temperature
(1, 12L. CGA 15281 is,iJ1contrastq much less sensitive to
changes in temperature and has an energy of activation of
just 19 kcal mol'1 (11). In our study, solvolytic
fragmentation of CGA 15281 occurred quickly within the
fruit. This burst of ethylene apparently enhanced
anthocyanin biosynthesis through the activation of the PAL
enzyme, but was diffused from the tissue prior to synthesis
of the ripening enzymes. Consequently, anthocyanin
biosynthesis was enhanced in apple fruits without loss of

postharvest integrity.

Literature Cited

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Russell. 1976. Kinetic studies of the thermal
decomposition of 2-chloroethylphosphonic acrd in
aqueous solution. Plant Physiol. 58:700-792.

 

2. Chalmers, D. J., J. D. Faragher and J. W. Raff. 1973.
Changes in anthocyanin synthesis as an index of
maturity in red apple varieties. g; Hort. Sci.

 

48:389-392.

3. Creasy, L. L. 1968. The role of low temperature in
anthocyanin synthesis in McIntosh apples. Proc.!NL

 

Soc. Hort. Sci. 93:716-724.

 

10.

ll.

12.

l3.

14.

59

Dilley, C. L., and D. R. Dilley. 1985. New technology
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Dilley, D. R. 1983. Assessing fruit maturity and
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Faragher, J. D. 1983. Temperature regulation of
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34(147):1291-1298.

Faragher, J.[L and R.Iu Brohier. 1984. Anthocyanin
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Faragher, J. D. and D. J. Chalmers. 1977. Regulation
of anthocyanin synthesis :hi apple skin. III.
Involvement of phenylalanine ammonia-lyase. Aust. J;
Plant Physiol. 4:133-141.

 

Faust, M. 1965. Physiology of anthocyanin development
in McIntosh apple. I. Participation of pentose-
phosphate pathway in anthocyanin development. Proc.
Am. Soc. Hort. Sci. 87:1-9.

 

Flore, J. A. and M. ukovac. 1982. Factors
influencing absorption of 1 C- (2- -chloroethy1)phosphonic
acid fur leaves of cherry. J; Amer. Soc. Hort. Sci.
107(6):965-968.

 

 

Olien, W. C. 1980. Ethephon-induced gummosis in sour
cherry (Prunus cerasus): Effect of gum on xylem
function and influence of temperature. Ph. D. Thesis,
Michigan State Univ., East Lansing.

 

Olien, W. C. and M. J. Bukovac. 1978. The effect of
temperature on rate of ethylene evolution from ethephon
and from ethephon-treated leaves of sour cherry. J;
Amer. Soc. Hort. Sci. 133:199—202.

 

Phillips, W. R. and P. A. Poapst. 1952. Storage of
apples. Can. Dept. Agr. Bul. 776.

 

Roberts, J. K. M., P. M. Ray, N. Wade-Jardetzky, and O.
Jardetzky. 1986. Estimation (HE cytoplasmic and
vacuolar pH in higher plant cells by P NMR. Nature
283:870-872.

15.

16.

17.

18.

60

Roberts, J. K. M., D. Wemmer, P. M. Ray and O.
Jardetzky. 1982. Regulation of cytoplasmic and
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experimental conditions. Plant Physiol.

69:1344-1347.

 

Sando, C. E. 1937. Coloring matters of Grimes Golden,
Jonathan and Stayman Winesap apples. J; Biol. Chem.

 

117:45-56.

Siegelman, H. W. and S. B. Hendricks. 1958.
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Tan, S. C. 1979. Relationships and interactions
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