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This is to certify that the

dissertation entitled

Prediction and Study of Bitter Pit on Apples Using

Mg2+ Induced Bitter Pit-Like Symptoms
presented by

Douglas M. Burmeister

has been accepted towards fulfillment
of the requirements for

 

 

Ph.D. degree in Horticulture

 

Major professor J

Date Jz’f; AIL/7’73

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

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TO AVOID FINES return on or betore date due.

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MSU is An Afiirmetive ActiorVEqual Opportunity institution

chS-DJ

PREDICTION AND STUDY OF BI'ITER PIT ON APPLES USING
Mg2+ INDUCED BITTER PIT -LIKE SYMPTOMS

By

Douglas M. Burmeister

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1993

ABSTRACT
PREDICTION AND STUDY OF BITTER PIT ON APPLES
USING Mg2+ INDUCED BITTER PIT-LIKE SYMPTOMS
By

Douglas M. Burmeister

Bitter pit is a physiological disorder affecting apples. Apple fruit vacuum
infiltrated with Mg2+ develop bitter pit-like symptoms [Mg2“ induced pits (MgIP)]
that are Similar to naturally occurring bitter pit. Addition of Ca2+ to the
infiltration media attenuates Mg2+ induced pit and the number of bitter pit-like
lesions induced by Mg“ is inversely related to the endogenous (native) Ca2+
concentration of individual fruits. MgIP was studied as a predictor of bitter pit
development in storage and as a system to study bitter pit initiation and
development.

Induction of MgIP on Northern Spy apples (Malus domestica Borkh.) by
infiltrating Mg2+ salt solutions into the fruits was positively correlated with bitter
pit that developed naturally during storage. The endogenous (native) fruit Ca2+
concentration was inversely related to the MgIP and to bitter pit development
following storage.

Golden Delicious apple fruit were infiltrated with various concentrations of
Ca2+ and Mg2+ with and without Ca“-affecting reagents or other cations.

Including Trifluoperazine with Mg“ increased pitting over Mgz+ alone.

Verapamil and nefedipine had no effect on MgIP or its attenuation by Ca“.
Cyclopiazonic acid attenuated MgIP. Ethyleneglycol-bis(B amino ethyl ether)-
N,N‘tetra acetic acid (EGTA), and 2,3,5-triiodobenzoic acid (TIBA) attenuated
MgIP. Cycloheximide inhibited Mg“ induced pit. Heating at 38°C prior to
infiltrating the fruits attenuated MgIP. Cations Ba“, La“, Co“, Sr2+ included at
20.0mM all completely arrested MgIP induced by 0.18M Mg“. K+ and Na+
partially inhibited MgIP. We have demonstrated that treatments affecting calcium
homeostasis or cellular metabolism can alter MgIP development. We conclude
that Mng may be useful in studying bitter pit initiation and development.

An experiment was conducted to determine if prestorage heat treatments
could attenuate bitter pit development on Northern Spy and superficial scald on
Red Delicious and Law Rome apples. Attenuation of physiological disorders by
heat treatment beyond that attainable by CA and low temperature was not

apparent. Heat treatments were effective in improving flesh firmness retention.

Dedicated to the memory of my Mom,

Helen C. Burmeister

iv

 

ACKNOWLEDGEMENTS

The author wishes to acknowledge, Dr. David Dilley for providing this
excellent opportunity to study under his direction. His enthusiasm and commitment

to science and friendship have been a never ending source of encouragement.

Special thanks to Dr.’s Randy Beaudry and Eric Hanson - Horticulture, Dr.
Mark Uebersax and Dr. Jerry Cash - Food Science and Human Nutrition, and Dr.
John Wilson - Biochemistry, who served on my guidance committee. Their
knowledge and expertise helped to make my experience in graduate school very

meaningful.

Also a special acknowledgement to everyone in the Horticulture Department,
especially those in the postharvest and vinticulture groups for making graduate school

the best time of my life.

Finally, thanks to my Dad, Gordon L. Burmeister for giving what it takes to

get through this. This is for you, Dad.

Guidance Committee:

The journal paper format was chosen for this thesis in accordance with
departmental and university regulations. The thesis is devided into two chapters in
which the first is accepted for publication in Postharvest Biology and Technology, the
second has been accepted for publication in the Journal of Agricultural and Food

Chemistry.

vi

TABLE OF CONTENTS

Page

LIST OF TABLES ............................................ ix-x

LIST OF FIGURES ........................................... x-xi

LITERATURE REVIEW ....................................... 1
Literature Cited .......................................... 17

CHAPTER 1:

Correlation of Bitter Pit on Northern Spy Apples with

Bitter Pit-Like Symptoms Induced by Mg2+ Salt Infiltration .............. 24
ABSTRACT ............................................. 25
INTRODUCTION ........................................ 25
MATERIALS AND METHODS ............................. 27
RESULTS AND DISCUSSION .............................. 29
The Relationship Between the Number of Bitter Pits
per Fruit and % Incidence of the Bitter Pit Disorder .............. 37
REFERENCES .......................................... 39

CHAPTER 2:

Characterization of Mg2+ Induced Bitter Pit-Like Symptoms on

Apples: A model System to Study Bitter Pit Initiation and

Development ................................................. 45
ABSTRACT ............................................. 46
INTRODUCTION ........................................ 47
EXPERIMENTAL ........................................ 49
RESULTS AND DISSCUSSION ............................. 51
Oz Dependency of MgIP .................................... 66
REFERENCES .......................................... 68
SUMMARY CONCLUSIONS AND FUTURE DIRECITON S ....... 77

vii

 

APPENDIX

Page
APPENDIX ONE:
Table of Means for Chapter 1 ............................... 82
APPENDIX TWO:
Additional figures for Chapter 1 .............................. 87
APPENDIX THREE:
The Effects of Prestorage Heat Treatments on Physiological
Disorder Development and Softening of Northern Spy, Red
Delicious and Law Rome Apples .................................. 96
INTRODUCTION ........................................ 96
MATERIALS AND METHODS ............................. 97
RESULTS AND DISCUSSION .............................. 98
REFERENCES ......................................... 105

viii

Table

II.

III.

IV.

VII.

LIST OF TABLES

Page
Chapter 2
The effects of Ca2+ and Ca2+ channel blockers on
mean number of Mg“ induced pits per fruit. .................... 53
The effect of cyclopiazonic acid (CPA) on Mg2+
induced pitting ............................................ 54
The effect of EGTA and TIBA on Mg2+ induced pit. .............. 56
The effect of protein synthesis inhibitors and
antibiotics on Mg2+ induced pitting. ........................... 58
The effect of heat treatment at 38°C prior to
Mg“ infiltration. ......................................... 60
The effect of heat treatment at 38°C following
Mg2+ infiltration. ......................................... 61
The effect of Na+ and K+ on Mg2+ induced pitting ................. 63
APPENDIX ONE

Parameters measured for 1990, early harvest. Each number
represents a minimum of 30 fruit. ............................. 82
Parameters measured for 1990, late harvest. Each number
represents an average of a minimum of 30 fruit. .................. 83
Parameters measured for fruit harvested August 22, 1991.
Each number is the average of 50 fruit .......................... 84
Parameters measured for fruit harvested Sept. 3, 1991.
Each number represents the mean of 50 fruit. .................... 85
Parameters measured for fruit harvested Sept. 11, 1991.
Each number represents an average of 50 fruit. ................... 86

LIST OF TABLES (cont)
APPENDIX THREE
Table Page

1. Effect of prestorage heat treatments on flesh firmness
and bitter pit on Northern Spy apples stored at 3°C for
8 months in purge mode. .................................. 100

2. Effect of prestorage heat treatments on flesh firmness
and superficial scald on Red Delicious apples stored at
3°C for 8 months in purge mode. ............................ 101

3. Effect of prestorage heat treatments on flesh firmness and
superficial scald on Law Rome apples stored at 3°C for 8
months in purge mode. .................................... 102

4. Effect of prestorage heat treatments on flesh firmness and
superficial scald on Law Rome apples stored at 1°C for 8
months in Quasi-static CA system. ........................... 103

5. Effect of prestorage heat treatments on flesh firmness and
superficial scald on Law Rome apples stored at 3°C for 8
months in Quasi-static CA system. ........................... 104

LIST OF FIGURES

Figure Page

Chapter 1

Correlation between MgIP/fruit at harvest and natural

bitter pit development on Northern Spy apples during

storage in air. Fruits were harvested 20d (0) and 10d (El)

prior to and at optimal maturity (A). Each data point

represents the average of 50 fruits. All regressions

were significant at "P<0.01. For 5°C storage, A) MgIP/fruit

vs % of fruits with bitter pit and B) MgIP/fruit vs bitter

pits/fruit and for 3°C storage, C) MgIP/fruit vs % of fruits

with bitter pit and D) MgIP/fruit vs bitter pits/fruit. .............. 32

The relationship between the number of bitter pits per
fruit and % incidence of bitter pit for Northern Spy

stored at 3°. ............................................. 38
Chapter 2
Dependency of MgIP on 02 concentration (%) .................... 67
APPENDIX TWO

Fig. 1. Plot of MgIP/Fruit vs % of fruit with bitter pit
that developed during storage at 5°C for 5 months. Data
are cumulative for both the 1990 and 1991 seasons.
For MgIP/fruit vs bitter pits/fruit that developed
in storage,Y= 1.9X + 3.3, r2: 0.31. For % of fruit with
MgIP vs % of fruit with bitter pit after storage,
Y= 0.92X + 14.5, r2: 0.51. ................................. 88

Plot of endogenous (native) calcium (pg g’l dwt.) vs
MgIP/fruit ............................................... 91

Plot of endogenous (native) calcium (pg g" dwt.) vs

bitter pits/fruit that developed in storage.

For 5 °C and 3°C Storage Y= 473.6’EXP(-0.02*X), r2: 0.49,

and Y= 502.0'EXP(-0.043), r2: 0.59, respectively ................ .93

Plot of % of fruit with MgIP vs % of fruit with bitter
pit after 3°C air and CA storage for 7 months. ................... 95

xi

LITERATURE REVIEW

Description of Bitter Pit.

Bitter pit is in a group of physiological disorders affecting apples and pears
known as corking disorders. These include bitter pit, corky core, cork spot, and
crinkle. Faust and Shear (1968) have reviewed corking disorders. Bitter pit is
characterized by sunken lesions that may develop on fruits as they mature on the
tree or after harvest during storage. The tissue below the skin in the pitted area
becomes discolored and dehydrated, resembling cork. When the lesions develop
while the fruit are on the tree, the disorder is sometimes referred to as tree pit.
Bitter pit is distinguished from other corking disorders in that lesions are mostly
located in the outer cortex just below the skin. The affected tissue is softer than
adjacent healthy tissue and it occurs at harvest or during storage.

Factors affecting bitter pit incidence.

The first reports of bitter pit were over 100 years ago by Jager (1889) as
cited by Faust and Shear (1968). The disorder has been of commercial concern
since the early 20th century and factors affecting its occurrence have been well
documented (Faust and Shear, 1968; Ferguson and Watkins, 1989). Environmental
factors and cultural practices that contribute to increased bitter pit incidence
include: light cropping, Ca2+ deficiency, excessive tree vigor, excessive N nutrition,
and moisture stress. Fruit harvested immature are also prone to the disorder.

Susceptibility to bitter pit varies among cultivars and geographically. A

cultivar can be considered susceptible in one region of a country or state and not

3

in another. For example, Smock and Neubert (1950) reported that Golden
Delicious when grown in the US. had no susceptibility. It is now known that
immature Golden Delicious can be prone to develop bitter pit and this is common
in the Pacific NW. Bitter pit has also been reported on Golden Delicious in
Australia, New Zealand, and South Africa (Ferguson and Watkins, 1989 and
references therein).

Mineral Content of the Fruit.

Of the factors contributing to bitter pit incidence, Ca2+ deficiency of the
fruit has emerged as the crucial factor. Delong (1936) was the first to observe
that low fruit Ca2+ levels correlated with bitter pit development in storage.
Garman and Mathis (1956) determined that spraying trees with Ca2+ salts to fruit
during development reduced bitter pit incidence. Since that time, the effects of
environment, cultural, and chemical treatments on the mineral content of apple
fruit in relation to bitter pit have been thoroughly investigated. In addition, high
levels of Mg2+ and/ or K+ may also predispose fruit to bitter pit. Perring (1986)
found that few apples were pitted in years when Ca2+ levels were high even if
their Mg2+ and K+ levels were high. When Ca2+ levels were marginal, more of
the bitter pit afflicted fruit had high K+ and Mg2+ levels. Thus, a direct
relationship between K“ or Mg2+ alone and bitter pit is often not detected
especially if native calcium levels are high, but high ratios of K‘“, and/or Mg“, to
Ca“ are associated with bitter pit frequently (Webster and Forsyth, 1979;

Hopfinger and Pooviah, 1979; Martin et al. 1960).

4

Studies attempting to relate levels of N, P, B, Na, Zn, and 01 to bitter pit
incidence have been reviewed in detail (Faust and Shear, 1968; Ferguson and
Watkins, 1989). No consistent relationships have been found indicating a critical
role for these elements in bitter pit.

Distribution of minerals within fruit.

Complicating the Ca2+/bitter pit relationship is the fact that mineral
constituents of apple fruit are not uniformly distributed (Wilkinson and Perring,
1961; Perring and Wilkinson 1965; Faust et a1. 1967). The Ca2+ concentration is
higher in the stem than in the calyx end of the fruit, higher in the core and skin
than in the cortex, and concentrations can vary around the fruit axis. Transverse
distribution of K” and Mg“ exhibits a similar pattern of distribution but there is
no longitudal gradient.

Considerable effort has been expended to relate changes in mineral
distribution during storage with bitter pit development. Translocation of Ca2+
from the core to the outer cortex of apples during storage has been reported
(Ferguson and Watkins, 1983; Bramlage et al. 1979; Perring, 1986). Information
concerning the movement of other ions is conflicting. In Spartan apples, an
accumulation of K’r P and Mg2+ in the inner cortical zones was recorded (Perring,
1984), whereas an outward movement of K+ and Mg2+ has been recorded for
Cox,s Orange Pippin (Ferguson and Watkins, 1983). In the latter case, the authors
surmised that ions may have been concentrated in the outer cortex by water losses

resulting in the appearance of migration. Perring (1989) proposed that the

5

pattern of redistribution of minerals may depend on variety. Perring (1986) and
Perring and Pearson (1986) associated an irregular transient withdrawal of Ca2+
from the mid- and outer-cortical regions during storage just prior to the
appearance of bitter pit lesions. This withdrawal of Ca“ was less pronounced in
samples that developed less bitter pit.

Prediction.

Another approach to reduce losses is to predict the potential of fruits to
develop bitter pit using mineral analysis. This is a useful commercial practice
(Ferguson and Watkins, 1989). Analysis of fruit Ca2+ or the ratio of K“, Mg“,
K+ + Mg“ to Ca2+ is commonly employed in these predictive methods. Fruits
are sampled from orchards prior to harvest and analyzed for Ca“. Lots of low
Ca“ fruits can be identified and marketed or processed prior to development of
bitter pit. Fruits from orchards with a high Ca2+ status can normally be safely
held for long durations in storage.

Although the relationship between bitter pit and Ca2+ is highly variable,
thresholds for Ca2+ where minimal or no bitter pit may be expected have been
established (Wills et a1. 1976). In one study, the correlation coefficient (r) ranged
from 0.37-0.90 for the Ca/bitter pit relationship (Wills et a1. 1976). Thresholds
vary depending on the tissue (eg. whole fruit, cortical plugs, or skin), variety,
growing region and season. Some of the variation in thresholds may reflect
different sampling and analysis procedures. Bitter pit incidence can vary greatly,

even at Ca2+ levels below threshold. This makes accurate prediction impossible.

6

Attempts to reduce variability by expressing Ca2+ on a different basis ( eg. mg Ca
per cell surface) or use of fractionating tissue into soluble and insoluble fractions
have been unsuccessful (Perring, 1986). Incorporation of other orchard factors
(eg. fruit size, tree age, cropping level) with mineral analysis is used to improve
prediction of bitter pit occurrence in storage (Holland, 1980). The practicality of
such predictive methods is limited due to the inherent variability of bitter pit
incidence within and among orchards, varieties and growing seasons (Wills et al.
1976).

For a predictive system to be practical, results must be known well enough
in advance of the harvest to allow time for management decisions. The
relationship of Ca2+ to bitter pit has been shown to be reliable up to 3 weeks
before harvest (Ferguson et al., 1979). Analysis of young fruitlet midway in the
growing season has also proven to be useful (Marcelle, 1989). However, fruit
Ca2+ analysis is expensive, time consuming, and requires specialized equipment.
Also, since there is a high variability among fruits within an orchard, large sample
sizes are necessary to assess bitter pit potential. Waller, (1980) suggested a
minimum of 30 fruit, preferably 80 to accurately assess calcium levels in orchards.
An alternative method of assessing fruit Ca levels would be useful.

Control.

Generally, attempts to control bitter pit under commercial conditions by

cultural practices such as soil Ca2+ application, summer pruning (to increase

fruit/leaf) ratio, and delayed harvesting, are only marginally effective (Perring,

7
1979; Boon, 1980). The primary methods employed in attempts to control the

disorder are: applying Ca2+ sprays to trees at intervals throughout the growing
season (Jackson, 1962; Cooper and Bangerth, 1976) using post-harvest dips or
drenches in Ca2+ solutions (Mason and Drought, 1975), or infiltrating Ca2+
solutions into fruit (Scott and Wills, 1979; Cooper and Bangerth, 1976). Control
of bitter pit by Ca2+ treatments is highly variable and not always complete
(Ferguson and Watkins, 1989).

Storage Conditions.

Bitter pit have been reduced by low temperature (Perring, 1986) and CA
storage (Hewett, 1984; Sharples and Johnson, 1987). High water loss during
storage is sometimes associated with increased bitter pit (Hewett, 1984; Scott and
Wills, 1979). The benefits of CA storage and postharvest Ca2+ treatments are
additive (Hewett, 1984; Webster and Forsyth, 1979; Johnson, 1979). CA storage
lowered the threshold Ca2+ level where no bitter pit was expected (Sharples and
Johnson, 1987). If ripening and metabolism are suppressed for long periods by low
0; storage, irreversible attenuation of bitter pit may result (Ferguson and
Watkins, 1989; Perring, 1986).

Structural and Biochemical Differences in Affected Tissue.

The site of bitter pit is associated with the ends of the vascular bundles
(Smock and Van Doren, 1937). Its structural development is distinguished from
other corking disorders by absence of abnormal growth of cells such as observed

in cork spot (Simons et al., 1971). It is believed this difference could be in the

8

timing of the disorder. Bitter pit occurs just before harvest, or during storage
when cells may be unable to initiate cell division (Faust and Shear, 1968). Pectin
proturbances into intercellular spaces have been observed (Simons, 1962). The
few structural studies of bitter pit development are of fruit that develop symptoms
while still on the tree (Ferguson and Watkins, 1989). A systematic study of
structural development of the anatomical aspects of bitter pit in storage has not
been accomplished.

During bitter pit development, there appears to be a general migration of
mineral and organic constituents into the pitted tissue. Pitted tissue is higher in
starch, glucose, and fructose, but low in sucrose and higher in N and pectin than
adjacent healthy tissue (Askew et al., 1960; Faust, et al., 1968a). Citric acid is the
main organic acid in pitted tissue, while malic acid was the predominant acid in
healthy tissue (Steenkamp and de Villiers, 1983; Faust and Shear, 1968). In
studies of Jonathan spot, another Ca2+ related disorder, Richmond et aL, (1964)
demonstrated movement of minerals into the affected area which was associated
with a higher level of total organic acids, mainly malic acid, in the affected tissue.

Consistently, the pitted zones of apple fruit are higher in Ca2+ and Mg2+
than non-pitted tissue (Steenkamp and de Villiers, 1983; Meyer et al. 1979). High
Mg2+/Ca2+ (Hopfinger and Poovaiah, 1979; Hopfinger et a1. 1984) have been
reported. Ford (1979) demonstrated that 45Ca2‘“ moved into the pitted area as
symptoms developed.

Cork spot, unlike bitter pit has a red color formation on the Skin above the

 

9

incipient lesion (Faust and Shear, 1968b). In etiological studies of cork spot,
ethylene production followed by a respiration rise were the first signs of the onset
of the disorder. This was followed by an increase protein synthesis, pectin
synthesis, and movement of ions into afflicted tissues. Acetate was the major
respiratory substrate utilized in the pitted tissue. They hypothesized the acetate
and ethylene were breakdown products of membrane fatty acids.

The mineral and biochemical changes documented for bitter pit and other
corking disorders are symptomatic of other diseased and mechanically injured
tissue. It is hypothesized that these differences between pitted and healthy tissue
are not related to the initiation, but are the result of the metabolic disturbance
and subsequent tissue breakdown (Ferguson and Watkins, 1989; Faust and Shear,
1968).

Calcium and Fruit Ripening.

Calcium’s influence on apple ripening is acknowledged, but not understood.
Fruit with a high Ca2+ content ripen at a slower rate than fruits with lower flesh
Ca2+ levels. This has been demonstrated by comparisons of respiration rates of
fruits within a range of Ca2+ contents (Faust and Shear 1972; Bramlage et aL,
1974), or between fruit with raised Ca2+ content by sprays during development on
the tree (Cooper and Bangerth 1976). Bramlage et aL (1974) suggested that Ca2+
reduced the rate, but not the timing of the respiratory climacteric in apples.

Red Delicious apples of low Ca+2 levels entered the ethylene climacteric

earlier than fruits with high Ca"2 (Tomala and Dilley, 1989). Avocado treated

 

10

with Ca+2 had both a depressed and a delayed ethylene climacteric (Tingwa and
Young, 1974). Ca+2 reduces ethylene production, and delays ripening and
subsequent softening of apples. This effect was mimicked by Sr”, but was not
seen with K”, or Mg+2 infiltration. Ida Red apples infiltrated with Ca+2 had
reduced ethylene, ACC content and ethylene forming enzyme (EFE) activity
(Tomala and Dilley, 1990). It is not known how Ca2+ or other cations affect
respiration, ethylene production and ripening. Ca+2 may diminish respiration by
decreasing membrane permeability thereby reducing the diffusion of respiratory
substrates from the vacuole to centers of metabolic activity in the cytoplasm
(Faust and Klein, 1974; Bangerth et a1. 1972; and Cooper and Bangerth 1976).
Direct effects of Ca2+ on respiration and ethylene production have not been
demonstrated (Ferguson, 1984). It has been suggested that calcium may be
required for some ethylene-dependent plant processes (Raz and Fluhr, 1992).
Calcium as a second messenger.

Ca2+ is as an essential element in plant nutrition, cell wall structure, and
membrane structure and function (Bangerth, 1979; Ferguson and Drobak, 1988;
Pooviah, 1988). Also Cams role as a second messenger mediating many plant
responses to external stimuli is now well accepted (Heplar and Wayne, 1985;
Poovaiah and Reddy, 1987). Ca2+ mediates plant responses through changes in
cytosolic Ca2+ concentration ([Ca2*]c,,). External stimuli (e.g. light, growth
regulators, gravity) imposes an action potential that results in the release of Ca2+

into the cytosol either by an influx through voltage-gated Ca2+ channels in the

11

plasma membrane, or by releasing Ca2+ from intracellular organelles such as the
vacuole, endoplasmic reticulum, chloroplast, and mitochondria (Wayne, 1993).
This increase in [Ca2+]cy, is transient and can be very localized (Cheek, 1989).
Ca2+ then binds to calmodulin, or other Ca2+ binding proteins which then leads to
a physiological response. This may involve the phosphorylation or .
dephosphorylation of certain enzymes and possibly gene expression.

Low [Ca2+]cy, is maintained against a concentration gradient (1000 fold)

between the cytosol and other cell compartments (vacuole and cell exterior. i;

 

Resting values for [Ca2+]cy, are between 0.1-1.0uM for all plants in which this has
been measured (Poovaiah and Reddy, 1987). Maintenance of low [Ca2+]cyt is
believed to be primarily accomplished by an active efflux of Ca2+ from the cytosol
via Mg2+ dependent Ca2+ activated ATPase located in the plasma membrane, and
by the similar uptake ATPases located in the membranes of the intracellular
organelles (Moore et aL 1984). The Ca“ ATPase of the plasma membrane is
believed to play a major role in plant homeostasis (Brisken, 1990; Carfoli, 1987).

In plants, measuring [Ca2+]cy, is problematic because of the presence of cell
walls, large vacuoles, chloroplasts, and high cellular turgor pressure. Harker and
Venis (1991) have measured intracellular and extracellular free calcium in apple
fruit using calcium-sensitive microelectrodes. Because of the highly vacuolated
nature of apple cells only one successful [Ca2+]cy, measurement was accomplished.
The [Ca2+],y, was verified to be 0.05uM. The apoplastic and vacuolar [Ca2+]

ranged from 0.02-1.3mM and 0.06-1.0mM, respectively. The total calcium in the

12

apple tissue was positively correlated with the apoplastic free calcium. Assuming
that 90% of the cell weight is vacuole, they calculated that the vacuole accounted
for 40% of the total calcium in apple tissue. As much as 60% is thought to be in
the cell wall and associated apoplastic space (Demarty er al. 1984). These results
concur with other measurements (Miller and Sanders, 1987; Felle, 1988).
Initiation and Development

Although mineral composition is a major factor, it is not considered the
cause of the bitter pit disorder. Early theories concerning the cause of bitter pit
include excessive transpiration, necrosis of immature cells filled with starch,
toxicity to sprays, and viral infection (Faust and Shear, 1968). The cause(s) and
mechanism of initiation and development of bitter pit are not known. Currently,
bitter pit is thought to result from a localized Ca2+ deficiency or mineral
imbalance, but there is no direct evidence for this (Ferguson and Watkins, 1989;
Perring, 1986). Mineral analysis has only been conducted at the tissue level.
Since it has not been possible to identify sites on fruit where pits might develop
and measurements of the relevant pools of Ca“ in apple fruit are scarce, the
etiology of bitter pit is unclear. Many theories have been proposed to account for
development of the disorder.

Simon (1978) proposed that exogenous water in the intercellular spaces of
the fruit may cause cells to swell. Because of Ca2+ deficiency they lose
permeability burst and dry out. This is not compatible with the observation that

high humidity conditions attenuate bitter pit (Hewett, 1984). Ferguson and

13

Watkins (1989) point out that the discrete nature of the disorder would require
localized changes in water relations.

Bangerth (1979) proposed that Mg“, K“, H+ and organic acid are
antagonistic to membrane function. That high levels of these ions compete for
Ca2+ binding sites thereby increasing membrane permeability and leading to loss
of cell function. Steenkamp and de Villiers (1983) suggested the role of Ca2+ in
preventing bitter pit was to protect membranes by chelating citric and oxalic acid
which they demonstrated dissolved the middle lamellae of apple fruit cells.

The suggestion of Perring (1986) that a localized Ca2+ deficiency resulting
from withdrawal of Ca2+ from the outer cortex accounts for development in
storage needs further study (Ferguson and Watkins, 1989). Due to the high
variation between samples, data are often expressed as percent of the total so
quantitative relationship between redistribution of minerals and bitter pit has not
been established (Fergusson and Watkins, 1989). Transverse sections used do not
account for longitudal movements of Ca2+. Storage conditions that would
attenuate bitter pit such as CA storage did not alter Ca2+ redistribution (Perring,
1984; Perring and Pearson, 1987).

Ferguson and Drobak, (1988), Ferguson and Watkins (1989), and Ferguson
(1990) have considered the various cellular compartments of Ca2+. Given: 1)
reliable relationships between Ca2+ and bitter pit are detectable; and 2) the major
compartments of Ca2+ in cells are the apoplast and vacuole (Harker and Venis,

1991), it would follow that one or both of these compartments may be critical to

14

bitter pit development. It is thought that the vacuole due to its low surface area
and large volume may provide the rapid transient rises of [Ca2+]cy, necessary for
cellular function (Ferguson, 1990). It has been proposed (Ferguson and Drobak,
1988) that the extracellular Ca“ directly accessible to the plasma membrane is
the critical pool involved in bitter pit development. This pool of extracellular
Ca2+ directly accessible to the plasma membrane is where exogenous Ca2+ is
suspected to exert its effect (Ferguson and Watkins, 1981). It is believed that
enough Ca2+ is usually present to maintain the structure and function of
membranes. However, during certain conditions that would require transient
rises in [Ca2+]cy, for signal transduction, the Ca2+ availability in this pool may
become limiting. The cell may then be unable to respond to external stimuli,
which in turn could lead to cellular disfunction (e.g. bitter pit). This could involve
the failure to maintain or to prevent certain metabolic events.

In this scheme, low native [Ca2+] is not always detrimental unless
exacerbating conditions such as drought or cold shock are present. Thus, a
particular Ca2+ level in fruit may be adequate under certain conditions, but may
not be adequate when there is an increased demand for Ca2+ for cellular activity
(eg. ripening). This proposal includes the role of Ca2+ as a second messenger and
accounts for several of the observations concerning bitter pit development in
apple including: 1) the variation in disorder development due to the factors
previously discussed; 2) that fruits of low total (native) Ca2+ content do not

necessarily develop the disorder; 3) fruits of varying maturities with similar Ca2+

15

contents differ in susceptibility; 4) irreversible attenuation can be affected by
application of CA; 5) increasing Ca“ contents artificially in the extracellular
space can attenuate bitter pit and affect intracellular events associated with
ripening and senescence (Glenn et al., 1988).

The apparent antagonism of bitter pit by Mg2+ has been previously
discussed. Fruit with bitter pit are often found to have high Mg“:Ca2+ ratios
(Perring, 1986). Mg2+ is a necessary cofactor for many enzymatic reactions.
There are examples of Ca/ Mg interactions in cellular functions. The intracellular
levels of Mg2+ are thought to be maintained in the mM range. Measurements of
Mg“ specific current (lug) in Paramecium indicate free Mg2+ concentrations were
in the range of 0.1-0.7mM. It is suggested that most of the intracellular Mg2+
exists in a bound form (White and Hartzell, 1989). Activation of the I”, was
found to be Ca2+ dependent (Preston, 1990). Sensitivity of Ca2+ channels to
blockage by extracellular Mg2+ has been demonstrated in N-methyl-D-Aspartate
(NMDA) receptor of nerve cells in vertebrates (Bumashev et aL, 1992). In apple
tissue, Harker et al. (1989) found that Mg2+ inhibited transport of Ca2+ in the
apoplast. Thus, Mg“ may have a cellular role for in bitter pit development.

Bitter pit-like symptoms (Mg2+ induced pit) have been induced on apples
by Mg“ by infiltration treatments whereas other divalent and monovalent cations
have not shown this effect (Conway and Sams, 1987; Hopfinger et aL, 1984;
Fallahi et aL, 1987). Researchers disagree as to whether Mg2+ induced pits are

the same as the natural bitter pit disorder. It has been suggested that the Mg2+

16

induced pits result from breakdown caused by Mg“ toxicity at the entry point into
the fruit (Ferguson and Watkins, 1989). Also, Mg“ is known to stimulate
polyphenol oxidase activity (an enzyme involved in the browning reactions
associated with the death of cells) in fruits vacuum infiltrated with Mg“
(Hopfinger et al., 1984). Fallahi et al., 1987 compared Mg“ induced pits to
natural bitter pit and concluded that induced pits lacked corky tissue. We have
demonstrated that these Mg“ induced pits are similar to naturally occurring bitter
pit (Burmeister and Dilley, 1991). Addition of Ca“ to the infiltration media
attenuates Mg“ induced pit and the number of bitter pit-like lesions induced by
Mg+2 is inversely related to the endogenous (native) Ca“ concentration of
individual fruits. Using these Mg“ induced bitter pit-like lesions, as a model for
bitter pit may provide new insight into the nature of the bitter pit disorder and

provide means to assess bitter pit potential.

17

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Ferguson, LB. 1984. Calcium in plant senescence and fruit ripening. Plant and
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Harker, RR. and Venis, M.A., 1991. Measurement of intracellular and

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20

Heplar, PK, and Wayne, RD. 1985. Calcium and plant development. Annu.
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Mason, J.L. and Drought, B.G. 1975. Penetration of calcium into Spartan apple
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Meyer, B.R., Peisach, M. and Kotze, W.A.G. 1979. Analysis of sound and pitted
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Miller Al, and Sanders, D. 1988. Depletion of cytosolic free calcium induced by
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Moore, AL, and Akerman, K.E.O. 1984. Calcium and plant organelles. Plant Cell
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21

Perring, MA. 1979. The effects of environment and cultural practices on calcium
concentration in the apple fruit. Comm. Soil Sci. Plant Anal. 10:279-293.

Perring, MA. 1984. Redistribution of minerals in apple fruit during storage.
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Acta Hort. 258:365-372.

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calcium content of appleszcalcium distribution in the fruit. J. Sci. Food
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Perring, M.A, and Pearson, K. 1987. Redistribution of minerals in apple fruit
during storage: the effect of Storage atmosphere on calcium concentration.
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The radial distribution of chemical constituents in apples, and its
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22

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23

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Chapter 1

Correlation of Bitter Pit on Northern Spy Apples with Bitter Pit-like Symptoms

Induced by Mg2+ Salt Infiltration.

24

25
ABSTRACT:

Induction of bitter pit-like symptoms (Mg“ induced pits [MgIP]) on
Northern Spy apples (Malus domestica Borkh.) by infiltrating Mg“ salt solutions
into the fruit was positively correlated with bitter pit that developed naturally
during storage. Fruit at harvest were infiltrated with 0.1M MgCl2 in 0.3M sorbitol
with 0.1% Tween 20 and placed at 20°C for 10 days ((1) after which the number of
MgIP was determined on individual fruits. A parallel sample of fruits from each
orchard was stored at 5°C in air (both years) and at 3°C in air or controlled
atmosphere (CA) storage in the second year. MgIP was positively correlated with
bitter pit development in storage for fruits harvested 20d and 10d before and at
optimal maturity for long term storage. The endogenous (native) fruit Ca“
concentration was inversely related to MgIP and to bitter pit development
following storage.

INTRODUCTION:

Bitter pit is a physiological disorder affecting apples. It is characterized by
sunken lesions that may develop on fruits as they mature on the tree or during
storage. The tissue below the skin in the pitted area becomes discolored and
dehydrated (Faust and Shear, 1968). Certain environmental and cultural
conditions contribute to increased bitter pit incidence including: light cropping,
Ca“ deficiency, excessive tree vigor, excessive N nutrition, and moisture stress.
Of these, calcium deficiency is the major factor that has been associated with

bitter pit (Delong 1936; Garman and Mathis, 1956; Ferguson and Watkins, 1989).

26

Low fruit Ca levels consistently correlate with high levels of bitter pit (Ferguson et
al., 1979; Wills et al., 1976; Waller, 1980). The primary methods employed in
attempts to control the disorder are: applying Ca“ sprays to trees at intervals
throughout the growing season, using post-harvest dips or drenches in Ca“
solutions, or infiltrating Ca“ solutions into fruit (Scott and Wills, 1979; Cooper
and Bangerth, 1976). Control of bitter pit by Ca“ treatments is highly variable
and not always complete.

Another approach employed to reduce losses from bitter pit is to use
predictive methods based upon mineral analysis to determine the fruits potential
to be afflicted with the disorder. This has been shown to be useful and is
practiced commercially (Ferguson and Watkins, 1989). Analysis of fruit Ca“ is
commonly employed for these predictive methods. Fruit are sampled from
orchards prior to harvest and analyzed for Ca. Orchards with low Ca fruits can
thereby be segregated for early marketing or the fruits can be processed prior to
development of the bitter pit disorder. Fruit from orchards with a high Ca“
status can normally be safely held for long durations in storage. Predictive systems
are in practice that use fruit Ca“ analysis alone, or in combination with other
orchard factors (eg. fruit size, tree age, cropping level), to predict bitter pit
occurrence in storage (Holland, 1980). The practicality of such predictive
methods is limited due to the inherent variability of bitter pit incidence within and
among orchards, varieties and growing seasons (Wills et al., 1976). Timeliness is

important. For any predictive system to be practical, the result must be known

27

well enough in advance of the harvest to allow time for management decisions.
The relationship of Ca“ to bitter pit has been shown to be reliable up to 3 weeks
before harvest (Ferguson et aL, 1979). Analysis of young fruitlets midway in the
growing season has also proven to be useful (Marcelle et al., 1989). However,
fruit Ca“ analysis is expensive, time consuming, and requires specialized
equipment. Also, because of the high variability among fruits within an orchard,
large sample sizes are necessary to assess bitter pit potential (Waller, 1980). An
alternative method of assessing fruit Ca levels would be useful. Researchers have
reported the induction of bitter pit-like symptoms (MgIP) on apples within 7d of
Mg“ treatment by infiltration (Conway and Sams, 1987; Hopfinger et al., 1984;
Fallahi et al., 1987). We have demonstrated that MgIP are similar to naturally
occurring bitter pit (Burmeister and Dilley, 1991). Addition of Ca“ to the
infiltration media attenuates MgIP and the number of MgIP is inversely related to
the endogenous (native) Ca“ concentration of individual fruits. If orchards of a
low Ca“ status have fruit more susceptible to MgIP then it may be possible to
assess bitter pit potential in a relatively quick and inexpensive manner. The
results reported herein are from a 2-year study correlating susceptibility to MgIP
with the occurrence of bitter pit in storage.
MATERIALS AND METHODS:

Orchards were selected that exhibited a range of bitter pit susceptibilities
based on orchard history. The number of orchards sampled, locations and harvest

dates varied between years. Orchards from the eastern, western, and

28

southwestern districts in Michigan were used in this study.

1990 Studies. Northern Spy and Red Delicious cultivars were employed.
Apples from 5 locations were harvested at ca. 1 week prior to, and from 11
locations at maturity (ca. 10% of fruits at > 0.2 pl 1‘1 internal ethylene
concentration). Samples of 10-15 fruit from each of 20 trees were randomly
selected at each location and were divided into two lots. One lot was vacuum
infiltrated (absolute pressure of 100mmHg for 2 min.) while submersed in 0.1M
MgC12 (50 fruit) in 0.3M sorbitol as osmoticum with 0.1% Tween 20 as surfactant.
The other lot was infiltrated with 0.3M sorbitol 0.1% Tween 20 which served as
the control (30 fruit). The fruits were held for 10d at 20° C, and the number of
MgIP lesions on individual fruits was recorded. None of the fruits infiltrated with
0.3M sorbitol with Tween 20 developed pits (data not shown). Parallel samples of
fruit not infiltrated were stored in perforated polyethylene bags in air at 5° C for 5
months after which the number of naturally-occurring bitter pit lesions on each
individual fruit was recorded. Individual fruits minus the core were analyzed for
calcium by atomic absorption spectrometry according to Tomala and Dilley,
(1989). Calcium data are expressed as ppm dwt (ug g‘1 dry wt).

1991 Studies. Only the Northern Spy cultivar was employed. Five orchards
were sampled ca. 20d, and 6 orchards at 10d prior to and at optimal maturity for
long term storage (Dilley and Dilley, 1985). Procedures were as previously
described except calcium analysis was conducted using a bulked sample consisting

of longitudinal sections (1 / 16 of each fruit excluding seeds) taken from 40 fruit

29

from each location/harvest combination. Storage treatments included air at 3°C,
and controlled atmosphere (CA) of 3.0% 02 + 3.0% C02 at 3°C for 6 months.
Fruit were held in 3°C air for 7d before CA was applied. Following storage fruit
were held for 7d at 20°C before assessment of bitter pit. These are the
commercially recommended storage conditions for Northern Spy in Michigan.

Data were analyzed by regression of MgIP vs bitter pit occurring during
storage. MgIP and bitter pit were assessed by determining the average number of
pits/fruit and the % of fruits with bitter pit. Calcium data were analyzed by
regression of endogenous (native) Ca vs mean number of MgIP, or % of fruit with
bitter pit that developed during storage.

RESULTS AND DISCUSSION:

For Red Delicious, incidence of MgIP and natural bitter pit development
were low for all locations sampled. This precluded development of prediction
equations for this variety. This may reflect a high calcium status of these blocks,
but this was not verified. Only the results for Northern Spy are presented.

1990. For the Northern Spy harvested early there was no correlation
between MgIP and natural bitter pit (see appendix 2, Fig. 1). For late harvest
1990, severity (bitter pits/fruit) and incidence (% of fruits with bitter pit) was
positively correlated with bitter pit after storage; Y= 1.76X + 1.2, r2: 0.69 and
Y= 6.7X + 20.0, r2= 0.57, respectively. In 1991, severity and incidence was
positively correlated with the severity of MgIP for all harvests for both the 5°C

(Fig. 1, A and B) and 3°C (Fig. 1 A and C) storage temperatures. Maturity of

30

fruit at harvest can be an important factor in subsequent development of bitter pit
(Ferguson and Watkins, 1989). We monitored fruit maturity in the 1991 season to
determine if maturity at harvest affected the correlation of bitter pit with MgIP.
An effect of harvest date on the correlation was not apparent (Fig. 1) but due to
weather conditions there was not as much variation in maturity between districts
as normally would be expected in a typical season (data not shown). Bitter pit
levels were higher for fruits stored in air at 5°C (Fig. 1, A and B) than stored in
air stored at 3°C (Fig. 1, C and D). Regressions for the 3°C CA yielded similar
prediction equations; severity vs severity and incidence were; Y= 0.32X + (-0.27),
r2: 0.69 and Y= 5.7X + (-5.6), r2: 0.73, respectively. All regressions were
significant at ”P<0.01. In most instances, severity and incidence of bitter pit were
lower in the 3°C CA treatment. We attribute this to a lower rate of metabolism
of fruits in CA. It has been demonstrated that postharvest conditions that slow
ripening and metabolism can reduce bitter pit levels (Perring, 1986).

The endogenous (native) Ca was inversely related to the average number
of MgIP/fruit and bitter pits/fruit that developed during storage (data not shown).
This type of relationship is considered typical when relating bitter pit to fruit
calcium levels. The exceptions to the relationship being fruit that are low in Ca
do not necessarily develop bitter pit (Ferguson and Watkins, 1989). In this study,
endogenous Ca levels below ca. 200 pg/ g dwt., a range of bitter pit levels can be
expected from 0 - >35 pits/ fruit depending on storage temperature (see appendix

2, Fig. 2 and 3, respectively).

31

Figure 1. Correlation between MgIP/fruit at harvest and
natural bitter pit development on Northern Spy apples
during storage in air. Fruit were harvested 20d (0) and
10d.(ED prior to and at optimal maturity (A). Each data
point represents the average of 50 fruits. All
regressions were significant at ”P<0.01. For 5°C
storage, A) MgIP/fruit vs % of fruits with bitter pit and
B) MgIP/fruit vs bitter pits/fruit and for 3°C storage,
C) MQIP/fruit vs % of fruits with bitter pit and D)
MgIP/fruit vs bitter pits/fruit.

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33

In utilizing MgIP to predict bitter pit some of the same problems
encountered with the use of Ca analysis are evident. The relationship between
bitter pit and Ca is highly variable. This makes accurate prediction of levels of
bitter pit impossible (Perring, 1986). However, threshold levels of Ca where
minimal or no bitter pit may be expected have been established (Wills et al.,
1976). Therefore, high variation does not necessarily preclude the use of MgIP as
a method to assess bitter pit potential. The correlation coefficient (r2) values
presented here are similar in magnitude to others used for bitter pit prediction
(Holland, 1980; Waller, 1980). Moreover, in no instance was a high level of MgIP
associated with a low level of bitter pit. For fruits stored at 3°C, our data indicate
when MgIP is below ca. 5 pits/fruit, bitter pit incidence was below 25% and
number of bitter pits/fruit 2.0 (Fig. 1, C and D). Prediction of % incidence of
bitter pit from % incidence of MgIP was highly correlated (r2 = 0.81) (see
appendix 2, Fig. 4).

Developing a practical prediction scheme for physiological disorders such
as bitter pit is problematic. Many issues need to be considered. We sampled
fruit from orchards with a wide range of tree ages, fruit maturities, and
environmental, and cultural conditions. We observed a good correlation between
MgIP and natural bitter pit occurrence up to 20d prior to the commercially
recommended harvest date. This time frame is desirable to provide lead time to
segregate fruit lots to be processed immediately from those to be processed after

long-term storage. A MgIP/bitter pit relationship could also be useful to indicate

34

the need for additional Ca“ treatment by postharvest dips. In order to determine
if the relationship we have found between the propensity of fruits for MgIP and
occurrence of bitter pit during storage may be generally applicable, experiments
need to be conducted with various cultivars under a variety of storage
temperature and atmosphere conditions. Postharvest handling and storage
practices can affect bitter pit occurrence. For example, a delay in establishing CA
conditions of greater than 7d promotes ripening during storage and can have a
detrimental effect by promoting the development of physiological disorders such
as bitter pit (Fica et al., 1985).

Assessment and prediction of bitter pit has been reviewed (Ferguson and
Watkins, 1989). It is unclear from the published reports whether low % incidence
of bitter pit represent a few fruit with light or severe symptoms or, if a high
average number of pits/fruit is the result of a few fruit with severe symptoms.
Any amount of pitting on an individual fruit destroys its fresh market (dessert)
value. In the case of the pie slice industry in Michigan both bitter pit incidence
and severity are considered important in regard to processing Northern Spy apples
(Geisler, 1992, personal communication, Coloma Fruit, Inc.). Fruits visually
estimated to have >15 pits after peeling are culled. Apple slices affected with
bitter pit can be removed during processing, or the pitted area peeled away, but
extremely high incidence of pitting slows the processing of apples destined to
secondary markets.

The correlation between MgIP and development of bitter pit in storage

35

extends our previous observations (Burmeister and Dilley, 1991) that MgIP is
similar to the bitter pit disorder. We believe that the method described has the
potential to be a reliable and inexpensive means to assess fruits according to their
potential for bitter pit. Can the assessment of MgIP at harvest be related to
bitter pit development potential in a practical manner? Counting the number of
fruit affected in a sample can be accomplished easily, rather than counting the
number of pits on each individual fruit. Perhaps an index that relates incidence
and severity such as used for superficial scald (Lurie et al., 1989) would be useful.

We have recently demonstrated that treatments affecting calcium
homeostasis (eg. calmodulin antagonists, Ca“ chelators) can alter MgIP
development (Burmeister and Dilley, 1993). We speculate that the MgIP
development and the bitter pit disorder may share common mechanisms. Calcium
is recognized to be a second messenger in plant cells in mediating cellular
metabolism (eg. calmodulin, and Ca-ATPase) (Heplar and Wayne, 1985). Models
have been proposed to explain how calcium might regulate cell metabolism
(Ferguson and Drobak, 1988; Poovaiah, 1988). The cytosolic concentrations of
free Ca“ and Mg“ are known to be maintained at the submicromolar and
millimolar range, respectively. Ferguson (1990) has suggested that depletion of
the extracellular pool of Ca“ results in loss of the cells’ capacity to respond to
external stimulus resulting in cellular dysfunctions (eg. bitter pit).

Apple tissue susceptible to bitter pit has been shown to be low in Ca“,

especially in relation to the Mg“ level (Garman and Mathis, 1956). Harker et aL,

36

(1989) found that Mg“ inhibits “Ca“ transport across discs of cortical flesh of
apple fruit. Gilroy et al., (1989) reported that high extracellular Mg“ and K+
concentrations resulted in rapid breakdown of Ca“ homeostasis of carrot
protoplasts. Sensitivity of Ca“ channels to blockage by extracellular Mg“ has
been demonstrated in animal systems (Bumashev et al., 1992). Infiltration of Ca“
in the extracellular space of apple fruit can attenuate bitter pit and affect
intracellular events associated with ripening and senescence (Glenn et al., 1988).
Combined with these observations, our data suggest a more specific role for Ca“
and Mg“ in bitter pit development. We speculate that extracellular Mg“
supplied by infiltration may affect the supply of Ca“ in the apoplast of apple fruit
influencing the cells ability to regulate cytosolic Ca“; either by perturbation of a
voltage-regulated Ca“ channel or displacing Ca“ from ionic binding sites in the
apoplast. This could interfere with the role of Ca“ as a second messenger
involving a Ca“-ATPase, or Ca“/calmodulin linked phosphorylation of an
enzyme, or a regulatory protein involved in cellular homeostasis, or metabolism.
We believe Mg“ exacerbates the potential for apple fruit to initiate the chain of

reactions involved in expressing this Ca“ related disorder.

37

The Relationship Between the Number of Bitter Pits per Fruit and % Incidence

of the Bitter Pit Disorder.

Interpretation of research data on bitter pit and how this may relate to the
practical significance of bitter pit in the commercial sector may yield different
opinions. Assessment of bitter pit and other fruit storage disorders is in question.
It is sometimes not clear from the published reports whether low % incidence of
bitter pit represent a few fruit with light or severe symptoms or, if a high number
of pits/fruit is the result of a few fruit with severe symptoms. Any amount of
pitting on an individual fruit destroys its fresh market (dessert) value. In the case
of processing apples as fresh slices for the bakery industry both bitter pit
incidence and severity are considered important (Geisler, personal
communication, Coloma Fruit, Inc. 1992). Fruits visually estimated to have > 15
pits after peeling are culled. Apple slices affected with bitter pit can be removed
during processing, or the pitted area pared away, but extremely high incidence of
pitting slows the processing of apples destined to the bakery industry. It is
important to have a basis to measure the bitter pit disorder for both commercial
and research purposes.

Meaningful assessment of bitter pit is important in terms of any prediction
scheme that might be employed. Counting the number of fruit affected in a
sample can be accomplished easily, rather than counting the number of pits on

each individual fruit. An numerical index that relates incidence and severity such

38
as used for superficial scald may be useful.

The relationship between number of pits per fruit and % incidence of ‘
bitter pit for Northern Spy stored at 3°C appears to be linear (Fig. 2). If this
relationship is proven to be consistent between seasons severity could be

predicted on the basis of incidence.

 

100

l:

“-30 a a
n: a
E Y = 16.4X + 0.78

550 r’= 0.93

I

t o

340

El

0.

3 a

one a o

I... o

0 . a

'8 o :‘

 

NUMBER OF FITS/FRUIT

Figure 2. The relationship between the number of bitter pits per fruit and %

incidence of bitter pit for Northern Spy stored at 3°C.

39
REFERENCES:

Burmeister, D.M. and Dilley, DR. 1991. Induction of bitter pit-like symptoms on
apples by infiltration with Mg“‘2 is attenuated by Ca”. Postharvest Biol.

Tech. 1:11-17.

Burmeister, D.M. and Dilley, DR. 1993. Characterization of Mg“ induced bitter
pit-like symptoms on apples: A model system to study bitter pit initiation

and development. J. Agr. Food Chem. in press.

Burnashev, N., Schoepfer, R., Monyer, H., Ruppersberg, J .P., Gunther, W.,
Seeburg, RH, and Sakman, B. 1992. Control by asparagine residues of
calcium permeability and magnesium blockade in N MDA receptor.

Science. 257:1415-1419.

Conway, W.S. and Sams, CE. 1987. The effects of postharvest infiltration of
calcium, magnesium, or strontium on decay, firmness, respiration, and

ethylene production in apples. J. Amer. Soc. Hort. Sci. 112:300-303.

Cooper, T. and Bangerth, F. 1976. The effect of Ca and Mg treatments on the
physiology, chemical composition and bitter pit development of Cox’s

Orange apples. Sci. Hort. 5:49-57.

40

Delong, WA 1936. Variations in the chief ash constituents of apples affected

with blotchy cork. Plant Physiol. 11:453-456.

Dilley, DR. and Dilley, CL. 1985. New technology for analyzing ethylene and
determining the onset of the ethylene climacteric of apples. Proc. of 4th

Natl. Controlled Atmosphere Res. Conf. 4:353-362.

Fallahi, E., Fighette, TL. and Wernz, J .G. 1987. Effects of dip and vacuum
infiltrations of various inorganic chemicals on postharvest quality of apple.

Commun. Soil Sci. Plant Anal. 18:1017-1029.

Faust, M. and Shear, CB. 1968. Corking disorders of apples: A physiological and

biochemical review. Bot. Rev. 34:441-469.

Ferguson LB. 1990. Calcium nutrition and cellular response. In: Calcium in Plant
Growth and Development. Proc. 13th Annual Riverside Symposium on
Plant Physiology. January 11-13, 1990. American Society of Plant

Physiologists, Rockville, MD, 20855.

Ferguson LB. and Drobak, B.K., 1988. Calcium and the regulation of plant grth

and senescence. HortScience 23: 262-268.

41
Ferguson I.B., Reid, MS. and Prasad, M. 1979. Calcium analysis and the

prediction of bitter pit in apple fruit. NZ. J. Agr. Res. 22:485-490.

Ferguson, LB. and Watkins, CB. 1989. Bitter pit in apple fruit. Hort. Rev. 289-
353.

Fica, J., Skrzynski, J ., and Dilley, DR. 1985. The effect of delayed cooling and
delayed application of CA storage of McIntosh apples under low and high
ethylene levels. Proc. 4th Natl. CA Res. Conf., Hort. Rept. No. 126, North

Carolina State Univ., Raleigh, NC. pp. 82-94.

Garman, P. and Mathis, W.T. 1956. Studies of mineral balance as related to
occurrence of Baldwin spot in Connecticut. Conn. Agr. Expt. Sta. Bull.

601:5-19.

Gilroy, S., Fricker, M., Blowers, D., Harvey, H., Collinge, M. and Trewavas, AJ.
1989. Calcium channels, cytosol calcium and plasma membrane
phospohorylation: an integrated calcium stat system. In: Plant Membrane
Transport: The Current Position. J Dainty (ed.) Department of Botany
University of Edinburgh. The King’s Buildings Mayfield Road, Edinburgh.

EH9 3JH, UK, pp.21S-224.

42
Glenn, G.M., Reddy, A.S.N. and Poovaiah, B.W. 1988. Effect of calcium on cell

wall structure, protein phosphorylation and protein profile in senescing

apples. Plant and Cell Physiology. 29:565-572.

Harker, F.R., Ferguson, LB. and Dromgoole, RI. 1989. Calcium ion transport
through tissue discs of cortical flesh of apple fruit. Physiol. Plant. 74:688-

694.

Heplar PK and Wayne, R0. 1985. Calcium and plant development. Annu. Rev.

Plant Physiol. 36:397-439.

Holland, DA 1980. The prediction of bitter pit. In: D. Atkinson, L.E. Jackson,
RD. Sharples and W.M. Waller (eds.) Mineral Nutrition of Fruit Trees.

Butterworths, London. pp. 380-81.

Hopfinger, J.A., Poovaiah, B.W. and Patterson, ME. 1984. Calcium and
magnesium interactions in browning of Golden Delicious Apples with bitter

pit. Sci. Hort. 232345-351.

Lurie, S., Maier, S. and Ben Arie, R. 1989. Preharvest ethephon sprays reduce

superficial scald of "Granny Smith" apples. HortScience 24:104-106.

43
Marcelle, R.D., Porreye, W., Goffings, G. and Herregods, M. 1989. Relationship

between fruit mineral composition and storage life of apples, cv. Jonagold.

Acta Hort. 258:373-378.

Perring, MA. 1986. Incidence of bitter pit in relation to the calcium content of

apples: problems and paradoxes, a review. J. Sci. Food Agr. 37:591-606.

Pooviah B.W. 1988. Molecular and cellular aspects of calcium action in plants.

HortScience 23: 267-271.

Scott, KJ. and Wills, R.B.H. 1979. Effects of vacuum and pressure infiltration of

calcium chloride and storage temperature on the incidence of bitter pit and

low temperature breakdown of apples. Aust. J. Agric. Res. 30:917-28.

Tomala, K. and Dilley, DR. 1989. Calcium content of McIntosh and Spartan
apples is influenced by the number of seeds per fruit. In: J .K. Fellman
(ed.). Proc. 5th Int. Controlled Atmosphere Research Conf., 14-16 June,

1989, Wenatchee, WA, Washington State University, Vol. 1, pp. 75-82.

Waller, W.M., 1980. Use of apple analysis. In: D. Atkinson, L.E. Jackson, RD.

Sharples and W.M. Waller (eds.) Mineral Nutrition of Fruit Trees.
Butterworths, London. pp. 383-394.

44
Wills, R.B.H., Scott, K.J. and Smale, PJ. 1976. Prediction of bitter pit with

calcium content of apple fruit. NZ. J. Agr. Res. 192513-519.

Chapter 2

Characterization of Mg“ Induced Bitter Pit-like Symptoms on Apples: A Model

System to Study Bitter Pit Initiation and Development.

45

46

ABSTRACT

Vacuum infiltration of MgCl2 solutions into intact apple fruits induces
bitter pit-like symptoms [Mg“ induced pits, (MgIP)]. Including Ca“ in the
infiltration media prevents MgIP. Golden Delicious apple fruit were infiltrated
with various concentrations of Ca“ and Mg“ with and without Ca“-affecting
reagents or other cations. Including trifluoperazine (TFP) with Mg“ increased
pitting over Mg“ alone. Verapamil and nefedipine had no effect on MgIP or its
attenuation by Ca“. Cyclopiazonic acid (CPA) attenuated MgIP. Ethyleneglycol-
bis(B-amino-ethyl-ether)-N,N ‘tetra acetic acid (EGTA), and 2,3,5-triiodobenzoic
acid (TIBA), attenuated MgIP. Cycloheximide and actinomycin D inhibited
MgIP, while puromycin had no effect. Heating fruits at 38°C prior to infiltrating
the fruits with MgCl2 attenuated MgIP. Cations Ba“, La“, Co“, Sr“ included at
20.0mM prevented MgIP (induced by 0.18M Mg“). Ca“ (3.0mM) included with
0.18M Mg“ inhibited MgIP 50%. K+ and Na” partially inhibited MgIP. We
have demonstrated that treatments affecting calcium homeostasis or cellular
metabolism can alter MgIP development. We conclude that MgIP may be a

useful tool to understanding natural bitter pit development.

Key words: apple, bitter pit, calcium, channel blocker, homeostasis

47
INTRODUCTION

Bitter pit is a corking disorder in apples characterized by sunken lesions
that develop just prior to harvest or during storage. The tissue below the skin in
the pitted area becomes discolored and dehydrated (Faust and Shear, 1968).
Susceptibility to bitter pit varies among cultivars and geographic regions.

Disorder incidence has been associated with environmental and cultural
conditions. Excessive tree vigor, light cropping, calcium deficiency, and moisture
stress are among the factors that predispose the fruits to bitter pit (Faust and
Shear, 1968; Perring, 1986; Ferguson and Watkins, 1989). Fruits which are
immature at harvest are also prone to develop bitter pit. The relationship
between elemental nutrition and bitter pit development has been studied
extensively (Garman and Mathis, 1956; Martin et al., 1960; Jackson, 1962; Cooper
and Bangerth, 1976). Fruits with bitter pit are generally low in Ca, especially in
relation to high Mg levels. Treating fruits with Ca“ reduces pitting, while
treatments with Mg“ increases the incidence of pitting.

Pitted tissue contains high concentrations of Ca“ and Mg“ (Garman and
Mathis, 1956; Hopfinger and Poovaiah, 1979; Askew et al., 1960; Meyer et al.,
1979). Ford (1979) demonstrated that 4’Ca“ moved into the pitted area as the
tissue symptoms developed. Pitted tissue and normal tissue differ in many organic
and mineral constituents (Faust and Shear, 1968). Studies of Jonathan spot,
Richmond et a1. ( 1964), another Ca“ related disorder, demonstrated movement of

minerals into the affected area and this was associated with a higher level of total

48

organic acids, mainly malic acid, in the affected tissue. It is hypothesized that
these differences between pitted and healthy tissue are not related to the
initiation, but are the result of the metabolic disturbance and subsequent tissue
breakdown (Ferguson and Watkins, 1989).

The cause and mechanism of initiation and development of bitter pit are
not known. Bitter pit is thought to result from a localized Ca“ deficiency or
mineral imbalance, but there is no direct evidence for this (Ferguson and
Watkins, 1989; Perring, 1986). Since it has not been possible to identify sites on
fruit where pits might develop, studies of bitter pit are normally conducted on
fruit tissue showing visible symptoms of the disorder.

Bitter pit-like lesions were induced on apples after Mg“ treatment
(Hopfinger et al., 1984; Conway and Sams, 1987; Fallahi et al., 1987; K. Tomala,
1988 in our laboratory, unpublished data). Mg“ induced pits were synonymous to
bitter pit as indicated by: MgIP is counteracted by including Ca“ in the
infiltration media, and pitting incidence was inversely related to the native fruit
Ca“ level (Burmeister and Dilley, 1991). Further, we have correlated
susceptibility to Mg“ induced pitting with bitter pit occurring in storage
(Burmeister and Dilley, in press). Here we have used MgIP as a model for
investigating the physiology and biochemistry of bitter pit initiation and

development.

49
EXPERIMENTAL

Experiments were conducted with Golden Delicious apples (Malus
domestica borkh.), harvested in 1991 from the Michigan State University
Clarksville Horticultural Experiment Station, Clarksville, MI. Fruit
(preclimacteric at harvest) were held in controlled atmosphere storage (3% C02
+ 1.5% 02 at 0°C) for approximately 7 months. Randomly selected blemish-free
fruits (6-9 cm diameter) were vacuum infiltrated with various solutions by
submersing them at an absolute pressure of 100mm Hg for 2 min. All solutions
contained 0.3M sorbitol as an isotonic osmoticum, and 0.1% Tween 20 as a
surfactant. Sorbitol/surfactant solutions were included as controls. Thirty-six
fruits were employed in each treatment. After infiltration, fruits were stored for
10 days in air at 20°C and the number of bitter pit-like lesions MgIP on individual
fruits were then recorded. Analysis of variance was used to test for main effects
and interactions, or treatment sum of squares was partitioned into single degree of
freedom contrasts as appropriate for each experiment (Little, 1981).

Various chemical agents known to affect Ca“ availability, transport,
binding, or action were investigated to learn how perturbations of [Ca“] might be
involved in bitter pit development. These included: EGTA, a chelator; TTBA, an
auxin/Ca“ transport inhibitor; verapamil (Vp) and nefedipine (Nf), Ca“ channel
blockers; trifluoperazine (TFP), a calmodulin antagonist; cyclopiazonic acid
(CPA), a Ca“-ATPase inhibitor; cycloheximide and puromycin, protein synthesis

inhibitors; actinomycin-D, an inhibitor of RNA synthesis and several cations.

50

Calcium channel blockers verapamil (Vp), nefedipine (Ni) and calmodulin
antagonist trifiuoperazine (TFP). Treatments were factorially arranged with 3
levels of Ca“ (0.0M, 0.01M, 0.02M), and 3 levels of Mg“ (0.0M, 0.04M, 0.18M),
alone or with Vp (100pM) or TFP (100pM). Fruits were infiltrated with 100pM
Nf alone, or with 0.18M Mg“. Nf was dissolved in dimethylsulfoxide (DMSO)
and added to the solutions resulting in a final concentration of 0.1% DMSO. All
controls contained 0.1% DMSO.

Q'clopiazonic Acid (CPA). Fruits were infiltrated with CPA (40pM) or
0.18M Mg“, or CPA (40pM) with Mg“.

2,3,5-triiodobenzoic acid (TIBA) and ethyleneglycol-bis(B amino ethyl
ether)-N,N,N‘,N‘-tetra acetic acid (EGTA). Fruits were treated with EGTA (100
pM), and TIBA (100 pM) alone or with 0.18M Mg“.

Protein synthesis inhibitors and antibiotics. Fruits were infiltrated with
0.18M Mg“ alone, or with cycloheximide (25 pg ml"), or puromycin (6.25 pg ml'
‘), or actinomycin D (25 pg ml").

Heat treatments. Experiment 1. Fruits were heated for 0, 1, 2 or 3 days at
38°C then infiltrated with 0.18M Mg“. During the heat treatments the fruits
were in perforated polyethylene bags to prevent desiccation. Experiment 2.
Fruits were infiltrated with 0.18M Mg“ and placed at 20°C for 7 days, or 3 days
at 38°C followed by 4 days at 20°C, or 3 days at 20°C then 3 days at 38°C and
then to 20°C for 7 days. Fruits were enclosed in polyethylene bags as in

experiment 1.

51

Divalent and monovalent cations. Fruits were infiltrated with 0.18M Mg“
alone or with 1.25mM, 2.5mM, 5.0mM, lOmM, or 20mM Ca“. Fruits were
infiltrated with either 0.18M Mg“, or 0.18M Mg“ + 20mM Ca“ alone, or with
La“, Sr“, Co“ or Ba“ at 20 mM as chlorides. Fruits were also infiltrated with
these cations alone as control. Fruits were infiltrated with 0.18M Mg“ alone or
including K+ or Na+ at 40 mM.

RESULTS AND DISCUSSION
Including TFP with Mg“ increased pitting over Mg“ alone, and Ca“ attenuated
MgIP in the treatments that included TFP (Table I). All main effects and
interactions were significant (P = 0.05) except for Mg“*TFP, and
Mg“"'Ca“*TFP interactions. TFP is a calmodulin antagonist of the
phenothiazine series. Cytosolic [Ca“] increased in carrot protoplasts treated with
TFP (Gilroy et al., 1987). Inhibitors of this class have induced bitter pit-like
symptoms (Fukumoto and Nagai, 1983). Results of experiments using TFP must
be interpreted with caution because these drugs are suspected to have general,
non-specific detergent properties (Personal communication, Dr. Ian Ferguson,
DSIR Auckland, NZ). However, in more recent studies of Ca“ fluxes across the
plasma membrane of Commelina commuis L., Siebers et al. (1990) concluded that
the effect of TFP was to mobilize membrane associated Ca“ and trigger release
of Ca“ from vesicles. They suggest that TFP induces Ca“ influx and/or inhibits
Ca“ efflux across the plasma membrane. No evidence of a detergent effect of

TFP was found. TFP treated plasma membrane-rich vesicles were still able to

52

import “Ca“ after being washed of excess TFP. Since Ca“ attenuates pitting
induced by TFP, we believe that TFP may act through specific binding rather than
by a detergent effect. However, binding of TFP is not specific to calmodulin.
There are many examples of TFP binding to other Ca“ related proteins such as
troponin C, S-100, and Ca“ activated phospholipid-dependent protein kinase
(Hartshome, 1985 and ref. therein). Roufogalis et a1. (1983) suggested that TFP
binds to the activated state of the Ca“ and Mg“ stimulated ATPase of
erythrocytes. Therefore, TFP may cause several effects on calcium-linked
metabolism rather than affecting a single event by binding to one specific site.

Vp and Nf are members of the dihydropyridine series of Ca“ channel
blockers and are believed to block voltage gated Ca“ channels from the inner
side of the plasma membrane; they enter the Ca“ channel while it is in the open
state (Carfoli, 1987). These drugs affect many plant systems (Heplar and Wayne,
1985). Vp at 100 pM reduced pitting induced by 0.18 M Mg“ (Table 1).
However, the main effects of Vp and Vp‘Mg“ interaction were only significant at
P = 0.1. Analysis of variance showed no other significant effects. Vp did not
affect Ca“ attenuation of MgIP. Another Ca“ channel blocker Nf at 100 pM
also did not significantly reduce pitting induced by 0.18M Mg“ (data not shown).
Similar experiments with Vp and Nf included at 500 pM showed no consistent
reduction in MgIP (data not shown). These results suggest that Ca“ entry into

the cell is not required for induction of pitting by Mg“.

53
Table I

The effects of Ca“ and Ca“ channel blockers on mean number of Mg“ induced

 

 

 

 

pits per fruit.
Mg“ Concn Car“ concn. M
(M) 0.0 0.01 0.2
Average number of MgIP per fruit

0.0 0.0 0.0 0.0

0.04 1.0 0.0 0.0

0.18 18.7 0.6 0.0
limpM TFP

0.0 4.6 1.0 0.0

0.04 3.8 0.2 1.0

0.18 22.7 3.5 1.0
1 M V r amil

0.0 0.0 0.0 0.0

0.04 1.7 0.2 0.2

0.18 12.0 0.1 0.4

54

CPA, an inhibitor of Ca“-ATPase significantly attenuated MgIP (Table II).

Including CPA (lOpM) (a Ca“-ATPase inhibitor) with Ca“ did not affect the

Ca“ attenuation of MgIP (data not shown). CPA is a specific inhibitor of Ca“-

ATPase and is believed to act by preventing the conformational change (E1 to

E2) of the enzyme that is necessary for Ca“ transport (Seidler et al. 1989). In

animal systems, CPA inhibits P-type calcium dependent ATPases of the

endoplasmic and sarcoplasmic reticulum that do not require calmodulin for

activation. CPA treated vesicles have a reduced rate of Ca“

Table II

The effect of cyc10piazonic acid (CPA) on Mg“ induced pitting.

 

 

Treatment Mean number of pits per fruit
0.18M Mg“ 14.5

0.18M Mg“+CPA (40pM) 6.4 vs 0.18M Mg"2
CONTROLS:

0.3M Sorbitol Alone 0.0

0.3M Sorbitol + CPA (40pM) 0.0

 

Sig.

8*.

‘Treatments were partitioned into single degree of freedom contrasts as indicated.

* = p50.05, " = ps0.01, *" = p50.001

55
efflux (Riley and Goeger, 1990).

EGTA at 100 pM significantly attenuated pitting induced by 0.18M Mg“
(Table III). EGTA is a specific chelator of Ca“ (Heplar and Wayne, 1985). Its
effect may have been exerted by sequestering Ca“ in the apoplast thereby
reducing the amount of Ca“ available to be transported across the plasmalemma.
TIBA at 100 pM also significantly reduced the amount of pitting induced by
0.18M Mg“ (Table III). TIBA has been demonstrated to decrease calcium
accumulation in apple fruits (Tomala and Dilley, 1989). Ca“ transport in plants
has been linked to the polar transport of auxin and the latter is known to be
inhibited by TIBA (dela Fuente and Leopold, 1973; Banelos et al., 1987). TTBA,
by blocking auxin efflux from the cell could prevent Ca“ entry into the cell

enhanced by 0.18M Mg“ treatment.

56
Table III

The effect of EGTA and TIBA on Mg“ induced pit.

 

 

Treatment Mean number of pits per fruit
0.18M Mg“ 215

0.18M Mg“ + EGTA (100pM) 6.8 vs 0.18M Mg“
0.18M Mg“ + TIBA (100pM) 4.1 vs 0.18M Mg“
CONTROLS:

EGTA (104M) Alone 0,0

TIBA (104M) ()5

0.3M Sorbitol 0.0

 

1see footnote to table 2.

Sig.

33*

33*

57
Cycloheximide at 25pM totally inhibited MgIP development (Table IV).

This was also found in another experiment with cycloheximide at lOpg ml" (data
not shown). Cycloheximide blocks protein synthesis by inhibiting aminoacyl
transferase in peptide bond formation in the ribosome. Puromycin at 6.25pg ml"
did not significantly reduce MgIP development. This antibiotic is also an inhibitor
of normal protein synthesis by causing the cell to produce an abnormal
polypeptide. The reason puromycin was less inhibitory than cycloheximide in
reducing MgIP may be because the concentration employed was too low.
Actinomycin-D at 25pg ml" significantly inhibited MgIP development (Table IV).
This antibiotic inhibits RNA synthesis by binding to DNA. Fruit of treatments
that included antibiotics had decay symptoms that were distinguishable from
MgIP. Collectively, the results with the antibiotics suggest that the induction of
pitting by Mg“ may involve mRNA and proteins synthesized de novo subsequent

to Mg“ treatment.

58
Table IV

The effect of protein synthesis inhibitors and antibiotics on Mg“ induced pitting.

 

 

'h‘eatment Mean number of pits per fruit Sig.l
0.18M Mg“ 25.8
0.18M Mg“ + Puromycin (6.25pg mi") 21.7 vs Mg“ Alone n.s.

0.18M Mg“ + Actinomycin D (25pg ml") 9.4 vs Mg“ Alone "I.

0.18M Mg“ + Cycloheximide (25pg ml") 0.0 vs Mg“ Alone “W

CONTROLS:

Puromycin (6.25pg n11") 0.0
Actinomycin D (25pg ml") 0.0
Cycloheximide (25pgml") 0.0
0.3m Sorbitol alone 0.0

 

1see footnote to table 2.

59

Heating apples at 38°C for 1 to 3 days prior to infiltrating them with Mg“
markedly reduced the amount of MgIP (Table V). Heating fruits immediately
after Mg“ infiltration more than doubled in number of pits that developed (Table
VI), whereas heat treatment applied 3 days following infiltration with Mg“ pitted
to the same degree as fruits not heated. Collectively, these data indicate that
heating fruits prior to subjecting them to the stress of Mg“ infiltration lessens
their susceptibility to MgIP but exacerbates pitting when applied immediately
after Mg“ infiltration. Exposure of plants and harvested plant organs to
temperatures in the range of 35 to 40°C can profoundly affect physiological and
biochemical processes during and subsequent to the heat stress. Alteration of
transcription and translation is a response common to all plants heated in the
range of 35 to 40°C and this is known as the heat shock (HS) response (Nagao et
al., 1986). Heating induces the formation of a complex family of heat shock
proteins (HSP) ranging in molecular weight from about 10 kD to nearly 100 kD
(Kimpel and Key, 1985). Prestorage heat treatments of apples have been found
to inhibit ripening (although not irreversibly), reduce the rate of subsequent
softening of apples, and attenuate storage disorders (Porritt and Lidster, 1978;
Klein and Lurie, 1992). We hypothesize that heating ameliorated MgIP as a

consequence of evoking the heat shock response.

60

Table V

The effect of heat treatment at 38°C prior to Mg“ infiltration.

 

 

Heat Treatment [Mg“] Mean number of pits per fruit Sig.1
0d 0.18M 8.4
1d 0.18M 1.9 vs 0d, 0.18M Mg“ m
2d 0.18M 3.7 vs 0d, 0.18M Mg“
3d 0.18M 2.9 vs 0d, 0.18M Mg“ "“
CONTROLS:
0d 0.00M 0.0
1d 0.00M 0.0
2d 0.00M 0.0
3d 0.00M 0.0

 

1see footnote to table 2.

61
Table VI

The effect of heat treatment at 38°C following Mg“ infiltration.

 

 

Heat Treatment [Mg“] Mean number of pits per fruit Sig.l
20°C Continuous 0.18M 18.3

3d Heat 20°C 0.18M 46.2 vs 20°C Continuous "“
3d 20°C 3d Heat 0.18M 21.0 vs 20°C Continuous n.s.
CONTROLS:

20°C Continuous 0.00M 0.0

3d Heat 20°C 0.00M 1.6

3d 20°C 3d Heat 0.00M 0.0

 

1see footnote to table 2.

62

A relatively low Ca“ concentration was found to counteract pitting
induced by Mg“ (Burmeister and Dilley, 1991). The concentration of Ca“
necessary to attenuate pitting induced by 0.18 M Mg“ by 50% was ~3.0mM
(data not shown). La“, Co“, Sr“ and Ba“ at 20.0mM all completely attenuated
Mg“ pitting induced by 0.18M Mg“ (data not shown). La“ is a Ca“ channel
blocker (Heplar and Wayne, 1985). La“ may not be able to cross plant
membranes (Thompson et al., 1973). It has been demonstrated to block turnover
of the phosphorylated intermediate of the Ca-ATPase in the microsomal fraction
of maize coleoptiles (Briars and Evans, 1989). C0“ is known to block Ca“
induced seed germination (Wayne and Heplar, 1984) and also to inhibit ethylene
production by inhibiting ACC oxidase (Kuai and Dilley, 1992). Ba“ and Sr“ ions
can often substitute for Ca“ in the binding of ligands such as membrane proteins
(Heplar and Wayne, 1985). Ca“ channels of charophytes do not transport Ba“
and only slowly transport Sr“. Our results suggest that these cations may act on
Ca“ binding sites in the extracellular space.

K" (40.0mM) and Na+ (40.mM) included with 0.18M Mg“ only partially
attenuated MgIP (Table VII). This is about twice the concentration of Ca“ that
completely inhibited the induction of MgIP by 0.18M Mg“. The effect of Na+
could be on the Ca“-Na+ antiport (Darnell et al., 1990). K+ has been shown to
stimulate ATPase activity in microsomal preparations of apple fruit (Lurie and

Ben-Arie, 1983).

63
Table VII

The effect of Na+ and K+ on Mg“ induced pitting.

 

Treatment Mean number of pits per fruit Sig.

 

0.18M Mg“ Alone 17.4
0-18M Mg“ + 0.04M 10 10.7 vs 0.18M Mg“ -
0.18M Mg“ + 0.04M Na+ 5.8 vs 0.18M Mg“

 

1see footnote to table 2.

64

We have demonstrated that we can alter the development of MgIP with
treatments that are known to affect Ca“ homeostasis and cellular metabolism.
Given the similarities between MgIP and bitter pit, our results may imply specific
roles for Ca“ and Mg“ in bitter pit initiation and development. Ca“ mediates
many responses in plants and animals (Carafoli, 1987; Heplar and Wayne, 1985).
The concentration of Ca“ in the cytosol ([Ca“],_.,,) is maintained in the
submicromolar range (Ferguson and Drobak, 1988; Poovaiah and Reddy, 1987;
Poovaiah, 1988) by the sequestering of calcium into organelles and export of Ca“
across the plasmalemma by ATPases. Extracellular signals give rise to transient
increases in [Ca“],,, either by release from cellular organelles, or by the opening
of specific Ca“ channels in the plasma membrane. [Ca“],,, affects cellular
processes by binding to enzymes, or to Ca“ binding proteins such as calmodulin.
Ferguson (1990) suggested that the critical pool of Ca“ involved in bitter pit
initiation and development is the extracellular compartment directly accessible to
the plasma membrane. Sufficient extracellular Ca“ would be needed for cells to
respond to environmental signals. Insufficient Ca“ would prevent cells from
responding and cause cell disfunction (e.g. bitter pit). This explanation may
account for the fact that fruits of low Ca may not develop bitter pit unless
conditions (eg. drought, excessive tree vigor, immaturity of fruit at harvest) trigger
the cellular response.

The role of Mg“ in bitter pit initiation is not understood. The

concentrations of Mg“ and K+ are generally high in relationship to calcium in

65
fruits with bitter pit (Perring, 1986). Harker et al., (1989) found that Mg“

inhibits 4’Ca“ transport across discs of cortical flesh of apple fruit. Our data
suggest that high levels of Mg“ in the extracellular space may be an important
factor in bitter pit initiation perhaps by preventing the influx of extracellular Ca“
to the cytoplasm via specific Ca“ channels.

Gilroy et al., (1989) reported that high extracellular Mg“ and K+
concentrations resulted in rapid breakdown of Ca“ homeostasis of carrot
protoplasts. There is evidence for a Ca“ / Mg“ antagonistic relationship in the
activation and inhibition of the Mg“ dependent Ca“ ATPases (Kawaski et aL,
1979; Kylin and Kahr, 1973; Vianna, 1975) in the microsomal fractions of plants
and animals. This seems to vary among species and tissues. In apple fruit, Lurie
and Ben-Arie (1983) found both Mg“ and, to a lesser degree, Ca“ inhibited
ATPase activity of the plasma membrane. We speculate that extracellular Mg“
supplied by infiltration could disrupt cellular homeostasis via the key enzyme(s)
that regulate intracellular Ca“. This in turn results in the chain of reactions that
result in MgIP. The high levels of Mg“ infiltrated into the extracellular space
presumably are akin to the high Mg“ levels often found in fruits with bitter pit.
We believe that the pitting symptoms induced by Mg“ infiltration are

physiologically synonymous with the bitter pit disorder.

66
Additional Experiment.

0, Dependency 0f MgIP. Controlled atmosphere storage is known to
reduce the incidence and severity of bitter pit in apple fruit (Perring, 1986). This
may be attributed to retardation of ripening and senescence development and/ or
the lower rate of metabolism at the lowered 02 level. If the effect of Mg“
infiltration in MgIP is a consequence of reducing the rate of Oz-dependent
metabolism, MgIP should be lowered as the O2 supply to the fruit becomes
limiting. The 02 dependency was determined for Golden Delicious apple fruit.

Golden Delicious apple fruit were harvested preclimacteric (ca. 10% of
fruits at >0.2pl 1" internal ethylene concentration) from the Clarksville Michigan
State University and held in air at 1°C for 4 weeks. A preliminary experiment
was conducted in which fruits were infiltrated with 0.0M, 0.09M, 0.18M, and
0.36M Mg“ in 0.4M sorbitol: 0.1% Tween 20 and stored at 20°C in air or 3.0%
C02 with 1.5% or 3.0% 02 for 1 month. More MgIP was found in air than at the
reduced 02 atmospheres but symptoms of fermentation damage were evident at
the reduced 02 levels (data not presented).

In a second experiment, fruits were infiltrated with 0.0M, 0.18M as before
and stored at 20°C ventilating with 2.5, 5.0, 7.5, 10.0, or 21.0% 02 for 12d. MgIP
increased for fruits infiltrated with 0.18M Mg“ as the 02 concentration increased
to 7.5% with no further increase (Fig 1). There was no apparent fermentation
damage evident at any of the 02 concentration employed. Since ripening changes

were not at issue in this experiment it appears that MgIP is an 02 dependent

67

metabolic process showing a Similar 02 saturation to respiration.

10

0.18M M 2* iNFlLTRATED
scans: 0.4M so BiTOL iNFiLTRATED

 

 

 

 

---D———-o——--a ----------- l—---D

[1 —‘l——T l i 1
2.5 5.0 7.5 1 0.0 1 2.5 1 5.0 17.5 20.0

02 CONCN. (96)

MEAN NUMBER OF Mg“ INDUCED PITS/ FRUIT

Fig 1. Dependency of MgIP on 02 concentration (%).

68
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77
SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS:

Bitter pit is a commercially important physiological disorder of apples.
The factors affecting its occurrence are well characterized . However, the causes
and mechanisms of bitter pit initiation and development, respectively are not
known. The disorder has been mainly associated with low fruit Ca and often
appears to be exacerbated by high Mg levels (Ferguson and Watkins, 1989).
Apple fruit vacuum infiltrated with isotonic sorbitol solutions containing Mg“
develop bitter pit-like symptoms [Mg“ induced pits (MgIP)]. We have
demonstrated that MgIP is similar to naturally occurring bitter pit. Addition of
Ca“ to the infiltration media attenuates MgIP and the number of lesions induced
by Mg“ is inversely related to the endogenous (native) Ca“ concentration of
individual fruits (Burmeister and Dilley, 1991). Studies were conducted using
MgIP as a means to assess bitter pit potential and as a model for bitter pit
initiation and development. Induction of MgIP on Northern Spy apples 10 days
after infiltrating 0.1M MgCl2 salt solutions into the fruits was positively correlated
with bitter pit that developed naturally in nontreated fruits during five and seven
months in storage at 5°C and 3°C , respectively. The endogenous (native) fruit
Ca“ concentration was inversely related to the number of pits induced by Mg“
and to bitter pit development following Storage.

Golden Delicious apple fruit were infiltrated with various concentrations of

Ca“ and Mg“ with and without Ca“-affecting reagents or other cations.

78

Including Triflquerazine (TFP), a calmodulin antagonist with Mg“ increased
pitting over Mg“ alone. Verapamil and nefedipine (calcium channel blockers)
had no effect on MgIP or its attenuation by Ca“. Cyclopiazonic acid (Ca“-
ATPase inhibitor) attenuated MgIP. Ethyleneglycol-bis(B amino ethyl ether)-N,N
,N‘,N‘-tetra acetic acid (EGTA), and 2,3,5-triiodobenzoic acid (TIBA) attenuated
MgIP. Cycloheximide (a protein synthesis inhibitor) inhibited Mg“ induced pit.
Heating at 38°C prior to infiltrating the fruits attenuated MgIP. Cations Ba“,
La“, Co“, Sr“ included at 0.02M all completely arrested MgIP induced by
0.18M Mg“. K+ and Na+ partially inhibited MgIP.

Many models could be proposed to explain MgIP development. From the
data presented it is difficult to draw specific conclusions. In general reagents, or
cations that would ameliorate influx of Ca“ across the plasma membrane or
sequester Ca“ attenuate MgIP. Cyclopiazonic acid would be expected to increase
MgIP by preventing Ca-ATPase from maintaining low cytosolic [Ca“]. Its
attenuation of MgIP could be the result of reducing membrane permeability. In
any case factors that would decrease [Ca“] attenuated MgIP. The multivalent
cations Ba“ and Sr“ and channel blockers Co“ and La“ substitute for Ca“ in
attenuation of MgIP. La“ is not believed to cross the plasma membrane.
Therefore Ca“ may be acting at the plasma membrane in preventing MgIP
development. Na" and K" may attenuate of MgIP by displacing Ca“ from ion
exchange sites in the cell wall. This would increase [Ca“] in the apoplastic

solution adjacent to the plasma membrane. Trifluoperazine may act directly on

79

calmodulin or calcium-modulated proteins interfering with cell function resulting
in MgIP. Cycloheximide may inhibit protein synthesis necessary for MgIP
development.

There is a paucity of information available concerning the roles of

extracellular Ca“ and Mg2

+ in the function of plant cells, especially bulky storage
organs such as apple fruit. In animal systems, recent evidence suggests that both
the extracellular and intracellular concentrations of Ca“ and Mg“ are important
in maintenance of cell Ca“ homeostasis. Intracellular Ca“ is suspected to be
modulated by both extra and intracellular [Mg“] in cardiac cells (White and
Hartzell, 1989). We speculate that high extracellular Mg“ may interfere with a
Ca“ channel in the plasma membrane. The presence of extracellular Ca“ may
alter membrane permeability to Mg“ thereby allowing the channel to be
inactivated. Little is known about Mg“ transport acrose cell membranes and a
regulatory role for Mg“ in cells. Our data indicate that an effect akin to this may
be occurring in development of MgIP. Thus, high [Mg“] in apple fruit may result
in inability of a Ca“ channel to close efficiently resulting in impairment of the
cells ability to regulate cytosolic [Ca“]. High levels of Mg“ and low levels of
Ca“ may in themselves not be detrimental, but when such a fruit is confronted
with a stress such as cold storage then the regulation of [Ca“] becomes critical.
This would lead to the event(s) (eg. protein synthesis) that result in bitter pit.

We have demonstrated that susceptibility to MgIP at harvest is correlated

with bitter pit development in storage and that treatments affecting calcium

80

homeostasis or cellular metabolism can alter MgIP development. These studies
indicate that extracellular Ca“ and Mg“ concentrations are important in bitter
pit development. This may be due to the influence of apoplastic Ca“ and Mg“
concentrations on maintenance of the cytosolic Ca“ concentration.

MgIP may be a useful to study bitter pit. Bitter pit is usually studied after
the disorder has already developed. MgIP can be induced in a matter of days
after Mg“ infiltration and this is attenuated with Ca“. MgIP may be useful as
model system to study bitter pit at the molecular level. Further research on the
effects of exogenous [Ca“] and [Mg“] on intracellular events such as protein
synthesis and phosphorylation following infiltration is warranted. Changes in
specific proteins could then be correlated with natural bitter pit development. If
changes in specific proteins occurred were identified then in vitro translation of
mRNA extracted at various times could be employed to detect mRNAs novel to
MgIP and bitter pit development.

The use of MgIP to study bitter pit is limited by the necessity of using
whole fruit. It would be desirable to use a tissue disc or cell culture to study the
effects of Ca“ and Mg“. Ideally, measurement of cytosolic Ca“ with fluorescent
dyes or Ca-selective electrodes would be measured. However, apple tissue is not
amiable to these techniques due to highly vacuolated cells and sensitivity to
wounding. Future effort should focus on development of an isolated system to
study the effects of Ca“ and Mg“ on apple fruit cells.

It is apparent that MgIP may be useful in understanding of the etiology of

81

bitter pit initiation and development as well as a practical means of assessing

bitter pit potential.

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APPENDIX TWO: Additional figures for Chapter 1.

87

88

Fig. 1. Plot of MgIP/Fruit vs % of fruit with bitter pit that developed during
storage at 5°C for 5 months. Data are cumulative for both the 1990 and 1991
seasons.

For MgIP/ fruit vs bitter pits / fruit that developed in storage, Y = 1.9X
+ 3.3, r2: 0.31. For % of fruit with MgIP vs % of fruit with bitter pit after
storage, Y= 0.92X + 14.5, r2: 0.51.

% OF FRUIT WITH BITTER PIT

89

 

  
  

 

100 .

j Y: 69.5*(1.0—EXP(-O.45*X))
90‘: r2: 0.54
806 V 00

I O
70: ‘
60%

50%
40%
30%

. 91 EARLY
20‘; 91 LATE
1O; 20 DAYS. 92

2 10 DAYS, 92

0.: . . . ‘ ' MATlIJRITY. 92

 

 

 

 

3 4 5 6 765310
MgIP/FRUIT

90

Fig. 2. Plot of endogenous (native) calcium (pg g“l dwt.) vs MgIP/fruit.

MgIP/FRUIT

91

 

14. Y: 54.2*EXP(—O.13*X)

12- 0 r2: 0.31
10; O

8‘ o

5-

      

O- _ Q30 0 O O 0
16C? boo T 240 ' 28o Y 326 1 E5336
ENDOGENOUS (NATIVE) Ca (pg g'1DWT.)

 

 

 

92

Fig. 3. Plot of endogenous (native) calcium (pg g" dwt.) vs bitter pits/fruit
that developed in storage. For 5 °C and 3°C storage Y = 473.6‘EXP(-0.02‘X),
r2= 0.49, and Y= 502.0’EXP(-0.043), r2: 0.59, respectively.

93

 

o u. 3°C STORAGE
o 5°C STORAGE

 

 

 

 

 

% OF FRUIT WITH BITTER PIT

 

166'266‘124ofi'2é6' Sio' Séso
ENDOGENOUS (NATIVE) Ca (ug g‘l DWT.)

94

Fig. 4. Plot of % of fruit with MgIP vs % of fruit with bitter pit after 3°C air
and CA storage for 7 months.

% OF FRUIT WITH BITTER PIT

95

 

‘ O AIR,3°C
A CA3°C

 

    

Y: 6.*EXP(O 07*X)

0.62
0.18

A A

 

 

oI1‘082'01310'4‘0'5'0'5'o‘7'o'ao
% OF FRUIT WITH MgIP

96
APPENDIX THREE: The Effects of Prestorage Heat Treatments on Physiological

Disorder Development and Softening of Northern Spy, Red Delicious and Law

Rome Apples.

INTRODUCTION

Exposure of plants and harvested plant organs to 35 to 40°C
temperatures profoundly affects physiological and biochemical processes during
and subsequent to the heat stress. Work in our laboratory as well as others has
shown heat treated tomatoes to be less sensitive to chilling injury (Picton and
Grierson, 1988; Lurie and Klein, 1990 and 1991; Burmeister et al., in
preparation). Other work with apples has shown an inhibition of ripening and a
decrease in softening rate of heat treated fruit (Maxie et a1. 1974; Porritt and
Lidster, 1978). In addition, superficial scald on apples has been shown to be
attenuated by prestorage heat treatments, although not completely (Lurie et al.,
1991; Klein and Lurie, 1992a). Interest in use of physical rather than chemical
means to control postharvest disorders has been prompted by concern of the
public over the safety of these chemicals (Klein and Lurie, 1992b).
Environmental Protection Agency registration of some fungicides, and
antioxidants may be withdrawn and the cost of re-registration of existing, or
registration of new chemicals is prohibitive. In addition, the storage and disposal
of the waste solution from drenching facilities is a problem. The objective of

this study was to determine if prestorage heat treatments in combination with

97

subsequent controlled atmosphere (CA) storage would attenuate physiological

disorder development on apples without sacrificing quality.

MATERIALS AND METHODS

Preclimacteric Northern Spy, Red Delicious, and Law Rome apples were
obtained from the Michigan State University Clarksville Experiment Station.
Purge CA system: Fruits (40-60) were randomized to fill into 20L CA chambers
by cultivar and held in air at 38°C for 0, 2, 3, or 4d and then placed in air at 1
or 3°C for the balance of 7d before establishing CA conditions. Chambers were
ventilated with 3.0% C02:1.5% 02, or 3.0% COZ:3.0% 02 at 75ml min".
Chambers left in air with loosely fitting lids served as controls. Each
atmosphere/heat treatment was done in triplicate. Quasi-static system: Law
Rome were randomized and loaded into CA chambers as before and held at
38°C for O, 1, 2, 3, or 4d then placed in air at 1 or 3°C for the balance of 7d
before establishing CA. CA was maintained employing a David Bishop
Instrument Oxystat II CA system. Fruit were stored for 8 months and examined
for disorders after a subsequent 7d period in air at 20°C. Northern Spy were
evaluated for bitter pit while, those of Red Delicious and Law Rome were
examined for superficial scald. Flesh firmness was determined with Effegi
penetrometer (11mm dia.) for 10 fruits upon removal from storage and after 7

days at 20°C in air.

98
RESULTS AND DISCUSSION

Purge system (3°C). Heating delayed softening of Northern Spy fruits
stored in air and with 3:3 atmosphere. The effect of heat was evident for fruits
on removal from storage and after holding them in air at 20°C for 7d (Table 1).
Attenuation of softening was significantly greater (P< 0.01) as the duration of
heating increased. Fruits stored at 1.5 02 retained the greatest firmness during
storage. Heating diminished the percentage of apples stored at 3:1.5 which
developed bitter pit but increased bitter pit on fruits stored at 3:3.

Red Delicious. Retention of flesh firmness and scald attenuation were
greatest in the 2d heat treatment at the 3.0:1.5 atmosphere (Table 2). However,
the effect of heat treatments on retention of flesh firmness and scald attenuation
for Red Delicious was not significant.

Law Rome. The effect of heat treatments on firmness upon removal
from storage was significant (P< 0.01), but not for fruits held for 7d at 20°C.
Heat treatments significantly increased scald for this cultivar.

Law Rome, quasi static system 1°and 3°C. Attenuation of softening for
heated fruit upon removal and after 7d at 20°C was significant for both 1° and
3°C storage temperatures (P<0.01). The effects of heat treatment on scald were
significant in both the 1°C and 3°C storage temperatures (P< 0.05 and P< 0.01,
respectively). Scald appeared to increase with duration of heating for fruit

stored at 1°C while decreasing for the 3°C storage temperature (Table 4 and S).

99

These results indicate that heat treatments to these varieties do attenuate
softening but may not be a viable alternative to control physiological storage
disorders in long term storage beyond appropriate application of low
temperature and CA. These results concur with those of Klein and Lurie
(1992a) who found no benefit on storage firmness following the heat treatment
of Granny Smith apples held in CA for 8 months. They did not evaluate these
long term CA fruit for physiological disorder development. In previous studies,
storage durations have been relatively short (Lurie et al., 1991). It may be that
positive effects of heat treatment on scald prevention may only be realized
following short storage durations or when application of appropriate CA
conditions is not feasible. Further investigations will be necessary to determine

conditions where prestorage heat treatment may be beneficial.

100

Table 1. Effect of prestorage heat treatments on flesh firmness and bitter pit
on Northern Spy apples stored at 3°C for 8 months in purge mode.

 

Flesh firmness (N)

 

 

951 2;! at 29°C

Days of heat Days Qf haat
Atmgsphgra Qd 2d 3;! 4d 991 2d 3d 4;!
(C0502)
Air 51.2 61.8 61.7 64.2 58.7 61.8 64.9 65.8
3.0:3.0 62.8 68.7 70.8 69.8 64.1 71.2 70.9 73.9
3.0:1.5 74.6 75.9 74.7 72.7 75.4 81.3 79.4 81.4

LSD0_05= 1.7 LSD0.05= 1.6

Bitter pit (%)

Air 17.6 10.7 12.8 12.8
3.0:3.0 0.7 0.0 9.0 6.8
3.0:1.5 11.1 1.5 2.6 0.0

IaSDon5 = 2.3

 

101

Table 2. Effect of prestorage heat treatments on flesh firmness and superficial
scald on Red Delicious apples stored at 3°C for 8 months in purge mode.

 

Flesh firmness (N)

 

 

0d ldJLZQiC
Days gr heat Days gr heat
mm 0d 2d 3d 4d a; 2g 3g 4g
(co,:o,)
Air 54.6 49.5 47.9 52.8 55.0 46.3 45.8 51.5

3.0:3.0 53.2 50.6 55.8 54.5 52.2 45.1 53.1 46.0

 

3.0:1.5 61.2 64.7 60.1 57.0 60.4 63.5 57.5 58.2

11813005 = 1.5 I-ISDOOS = 1.8

 

WW

Air 51.7 57.4 55.8 52.8
3.0:3.0 47.2 60.7 60.1 49.2
3.0:l.5 39.4 18.2 32.1 32.1

IJSDofls = 4.3

 

102

Table 3. Effect of prestorage heat treatments on flesh firmness and superficial
scald on Law Rome apples stored at 3°C for 8 months in purge mode.

Flesh fimnestm

 

 

SM 7 2 °
Days of hgt Days of heal;
Atmosphera on 2d 3d 4d Lid—M
(COzzOZ)
Air 56.9 67.8 73.7 70.1 52.0 59.7 71.6 66.1

3.0:3.0 65.6 71.7 73.5 73.0 65.5 67.8 64.6 60.5
3.0:1.5 75.4 74.6 73.3 72.4 67.2 73.3 67.2 67.7

11813005 = 2. 1 11813005 = 2.6

 

 

52W

AIR 75.7 69.9 74.4 80.1
3.0:3.0 16.2 25.6 37.0 34.0
3.0:1.5 10.7 21.3 16.4 27.4

I‘SDODS = 3.8

 

103

Table 4. Effect of prestorage heat treatments on flesh firmness and superficial
scald on Law Rome apples stored at 1°C for 8 months in quasi-static CA system.

 

Flesh firmness (N)

 

 

 

SM M
Days 9f hgat mm!
mm 0d 16 2d 3d 4d 1 2 4
(C050,)
Air 52.9 57.2 61.9 66.7 66.5 51.5 52.9 60.4 64.5 65.1
3.0:3.0 59.1 58.2 73.5 73.0 66.8 54.2 54.3 60.9 62.3 63.4
3.0215 59.7 60.4 73.3 72.4 63.2 57.5 57.1 59.2 59.8 61.8
I’SDO.05 = 1. 3 1.813005 = 1.7
1 o f
Air 72.0 72.6 75.1 71.2 80.0
3.03.0 34.3 45.4 47.6 58.1 54.7
3.0215 20.9 41.4 49.3 41.3 43.8

IJSDQOS = 5.3

 

104

Table 5. Effect of prestorage heat treatments on flesh firmness and superficial
scald on Law Rome apples stored at 3°C for 8 months in quasi-static CA system.

 

Atmgsphere
(C02202)

Flesh firmness (N)

m
Days Qf beat
Ed 1d 2d 3d 4d

 

7d gt29°g
h
0d 141 2d 3d 4d

 

 

AIR

3.0:3.0

3.0:1.5

44.0 47.9 53.6 57.3 56.4
49.5 53.2 53.1 54.2 55.8
53.5 53.4 53.8 55.5 57.5

14813005 = 1 .5

42.3 47.7 46.6 55.3 50.9
48.1 48.3 51.2 52.3 51.5
50.7 50.8 50.8 52.6 54.1

Lsnm= 1.2

 

Air

3.0:3.0

3.0:1.5

Scald (% Qf surfgg‘; area)
78.5 66.1 68.3 81.6 73.5

81.0 81.8 63.3 76.0 82.7
77.7 72.6 65.0 64.4 74.8

LSDQw = 2. 8

 

 

105
REFERENCES:

Burmeister, D.M., Dilley, D.R., Jianping, K., McNulty, D., Poneleitt, L.S., and
Walker, I. 1993 Postharvest heat treatments attenuate chilling-injury and
increase uniformity of ripening of tomatoes. Post Harvest Biol. and Tech. in
preparation.

Klein, J.D., and Lurie, S. 1992a. Prestorage heating of apple fruit for enhanced post-
harvest quality: Interaction of time and temperature. HortScience 27 :326-328.

Klein, J.D., and Lurie, S. 1992b. Heat treatments for improved postharvest quality of
horticultural crops. HortTechnology, 2(3):3l6-320.

Lurie, S., and Klein, JD. 1990. Heat treatment of apples: Differential effects on
physiology and biochemistry. Physiol Plant. 78:181-186.

Lurie, S., and Klein, JD. 1991. Acquisition if low-temperature tolerance in tomatoes
by exposure to high-temperature stress. J. Amer. Soc. Hort. Sci. 116: 1007- 1012.

Lurie, S., Klein, J.D., and Ben-Arie, R. 1991. Prestorage heat treatment delays

development of superficial scald on ’Granny Smith’ apples. HortScience 26: 166-
167.

Maxie, E., Mitchell, 6., Sommer, N., Snyder, G. and Rae, H. 1974. Effects of

elevated temperature on ripening of ’bartlett’ pear. Pyrus communis L. J. Amer.
Soc. Hort. Sci. 99:344-349.

Picton, S., and Grierson, D. 1988. Inhibition of expression of tomato ripening genes at
high temperature. Plant Cell and Environment. 11:265-272.

Porritt, S.W. , and Lidster, RD. 1978. The effect of prestorage heating on ripening and
senescence of apples during cold storage. J. Amer. Soc. Hort. Sci. 103:584-587.

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