Chillinginjuryinmangoes

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Chilling injury in mangoes
Article in Wageningen Agricultural University Papers · October 2005
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Loay Abd Ellatif Taha Arafat

Promotor:
Prof. Dr. O. van Kooten
Hoogleraar Tuinbouwproductieketens
Wageningen Universiteit, Nederland
Co-promotor:
Dr. Jeremy Harbinson
Universitair docent Tuinbouwproductieketens
Wageningen Universiteit, Nederland
Beoordelingscommissie:
Prof. Dr H.J. Wichers (A&F)
Prof. Dr Ir P.C. Struik (Wageningen Universiteit)
Dr L.M.M. Tijskens
Dr E. J. Woltering (A&F)
Dit onderzoek is uitgevoerd binnen de onderzoekschool Production Ecology &

Resource Conservation
Loay Abd Ellatif Taha Arafat
Proefschrift
ter verkrijging van de graad van doctor

op gezag van de rector magnificus
van Wageningen Universiteit
Prof. Dr M. J. Kropff,
in het openbaar te verdedigen
op dinsdag 4 oktober 2005
des namiddags om half twee in de Aula.
Loay Arafat (2005). Chilling injury in mangoes. Ph.D. Thesis. Wageningen University.
With references and summary in English, Dutch and Arabic.
Horticultural Production Chains Group, Department of Plant Sciences, Wageningen

University, Marijkeweg 22 (building number 527), 6709 PG Wageningen, The
Netherlands
ISBN: 90-8504-309-3
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Content
Chapter 1 General introduction 9
Chapter 2 Chilling injury and the balance between active oxygen species 23
and the scavenging capacity: a review
Chapter 3 Chlorophyll fluorescence assessment of chilling injury in ‘Zibdia’ 53

and ‘Hindi Be-Sennara’ mango varieties
Chapter 4 Changes in the concentration of water and lipid-soluble 83

antioxidants during cold storage of Zibdia’ and ‘Hindi Be-Sennara’
mango fruits
Chapter 5 Antioxidant enzymes and oxidative stress in mangoes 119
Chapter 6 On the Ethylene production, respiration rate, firmness and ion 163
leakage in mango fruits
Chapter 7 Final comment 191
Summary 207
Samenvatting 213
Arabic summary 219
Acknowledgement 223
Curriculum Vitae 225
Chapter 1
General Introduction
Chapter 1
Preamble
Mangoes are a popular, nutritional tropical fruit, which are now one of the
most important fruits crops in tropical and subtropical areas of the world. They
originated in India, were they have been cultivated for more than 4000 years.
Beginning in the16th Century, mangoes were gradually distributed from India to
other tropical countries in Asia such as the Philippines, Indonesia, China and
Thailand. They were also spread to the Americas in the 18th Century, and from
western Mexico they were carried to Hawaii in the early 19th century. The first
recorded introduction to Florida was at Cape Sable in 1833. Mango was introduced
to Egypt in the 18th Century from Ceylon by the Egyptian leader Ahmed Orabi upon
his release from internment. Many cultivars grown in Egypt today, such as ‘Zibdia’
and ‘Hindi Be-Sennara’ date form this time (Ibrahim and Khalif, 1999) Figure 1.
Increasing commercial acreage, and improved handling shipping
procedures are expected to increase the global market penetration of the fruits.
Currently, the major producers include India, Pakistan, Indonesia, Mexico, Brazil,
and the Philippines. Other important producers are Australia, South Africa, Egypt
and U.S (Crane and Campbell, 1994).
China
USA 16th
Mexico 18th India
Egypt
16th
18th
Philippines
Hawaii 16th
19th
Ceylon
16th
Indonesia
Malaysia 16th
16th
Figure1. The spread of mango from the original region of cultivation (India) round the world
Botanical and taxonomical of mango
10
The mango tree is a medium to large (9.1 to 30.5 m) evergreen tree with a
symmetrical, round canopy that ranges from low and dense to upright and open
(Figure 2). Leaves are alternately arranged, lanceolate, 15 to 40 cm in length, and
leathery in texture. Their colour is pinkish amber or pale green-coloured when
young, becoming dark green at maturity (Figure 3). The inflorescence is a many
branched panicle borne at the end of the shoot, it is 6 to 40 cm in length and
possesses from 550 to more than 4000 flowers. These are small, pinkish-white,
and mostly staminate (Figure.4). The fruits are classified as drupes and they vary in
shape (nearly round, oval, ovoid-oblong), size (from few 100s of grams to more
than 2.5 Kg), and colour (greenish, greenish-yellow, yellow, red, orange, or purple)
depending upon the variety. The skin is smooth and leathery and surrounds a
fleshy, pale-yellow to deep-orange edible pulp. The fruits possess a signal large,
flattened, kidney-shaped seed that is enclosed in a woody husk. Although the fruits
will ripen on the tree, commercially they are usually picked when firm and green for
shipment to the market. The crop is considered mature when the shoulder of the
fruit broadens (or fills out) and some fruits on the tree have begun to change colour
from green to yellow (e.g. Pride, Irwin and Tommy Atkins, ) (Litz, 1997). Prior to this
external colour break, the fruit is considered mature when the flesh near the seed
changes colour from white to yellow (Figure 5). Mango is harvested in three
maturity stages: immature (no shoulder development and of poor quality), half-
mature (the shoulder has developed to reach the same level as the stem insertion
point on the fruit) and full mature fruits (the shoulder has filled out and extends to
beyond the stem insertion point; this is the optimum quality) (Majeed and Jeffery,
2002; Mitra and Baldwin, 1997). Taxonomically, mangoes belong to the genus
Mangifera. It is one of the 73 genera, which include around 850 species, of the
family Anacardiaceae in the order Sapindales.
11
Chapter 1
Figure 2. Tree Figure 3. Different leaves maturity
Figure 4. Flower cluster with round 5000 flowers
Figure 5. Different size and colure of mango fruits which are related to different species.
12
The 69 Mangifera species that have so far been recognized are restricted to
tropical Asia.
Kingdom: Plantae (Plants)
Subkingdom: Tracheobionta (Vascular plants)
Superdivision: Spermatophyta (Seed plants)
Division: Magnoliophyta (Flowering plants)
Class: Magnoliopsida (Dicotyledons)
Subclass: Rosidae
Order: Sapindales
Family: Anacardiaceae (Sumac family)
Genus: Mangifera L.
Species: Mangifera indica L.
The Annual Global and Egyptian production of mangoes
Global production
Globally, mango production is increasing. In 2003, over 25 million tonnes of

mangoes were produced from about 90 countries. Asia was the main producer with
76.9% of the total world production, followed by the Americas with 13.8%, Africa
with 9% and less than 1% for Oceania (Table 1).
Table1. Distribution of world production during four years by continents (million

tonnes)(Source: www.fao.org)
Continent 2000 2001 2002 2003

Asia 18.65 18.66 19.95 19.06
America 2.51 2.53 2.49 2.50
Africa 2.50 2.64 2.62 2.63
Oceania 0.05 0.04 0.03 0.03
Total 23.70 23.87 25.09 24.22
Nine countries together produced 83% of the total mango production in 2003 (Table
2), of which India was the most important (10 million tonnes) followed by: China,
Thailand, Mexico, Pakistan, Indonesia, Philippines, Nigeria and Brazil.
13
Chapter 1
Table 2. The total production of main mango producing countries during the four year
period from 2000 to 2003 (Source www.fao.org)
Production (103t)
Countries 2000 2001 2002 2003 2004
India 10500 10240 10640 10500 10800
China 5211 3273 3513 3413 3622
Thailand 1633 1700 1750 1750 1750
Mexico 1559 155 1523 1503 1503
Pakistan 990 1037 1036 1036 1072
Indonesia 876 923 1403 731 800
Philippines 848 881 956 890 890
Nigeria 730 730 730 730 730
Brazil 538 782 842 845 845
Egyptian production:
The total mango production of Egypt was 197 million tonnes in 1997. The majority
of mango production (97.5% =192 million tonnes) is produced from by the Nile
valley provinces where most of the production area is concentrated in the provinces
of Sharkia, Ismaillia, Gisa, Fayuom, Qena and Nobaria, which produced
respectively 75, 37, 36, 17, 6 and 4.5 million tonnes each (Mamdouh, 1997) (Figure
6). The most important mango cultivars in the Egyptian market are, Hindi Be-
Sennara, Pairi, Tymour, Ewas, Zibida and langara. Mangoes are the sixth most
important fruit in the Egyptian market after citrus, grapes, olives, apples, and
banana (Mamdouh, 1997).
14
Figure 6. Distribution mango in 6 major provinces in Egypt.
Recently, according to the FAO organization (www.fao.com) (Figures 7 and 8).

the total harvesting area and production increased last four year (2000-2004)
50000
Total area harvesting (Ha)
A
40000
30000
20000
10000
0
1997 1998 1999 2000 2001 2002 2003 2004
Figure 7. Egyptian total area harvesting during last four years (1997to 2004)
15
Chapter 1
400
Total production (103t)
B
300
200
100
0
1997 1998 1999 2000 2001 2002 2003 2004
Figure 8. Total production 103 t during four years (2000 to 2004)
Mango market
The European Union mango market has developed greatly between 2000 and
2003, increasing by almost 10 million tonnes, and the growth in Mango
consumption is expected to continue to grow. The fruit is imported from many
countries, with Brazil being the main supplier with 33% of EU market in the year
2000 (Saúco, 2002). The increase in imports coupled with improved transport and
storage techniques has lead to the year round availability of mango in Europe but
the down-side for the producer is a lower price (Table 3). Egypt exports mangoes to
Europe, with England and France being the main importers (Information and
Decision Support Center 2003) However the Egyptian market also extends to Arab
countries such as Dubai, Saudi Arabia, Bahrain, and other Gulf countries. In order
to meet the growing demands from the Arab Gulf states and the EU the Egyptian
Agricultural ministry plans to increase both mango production and the total
plantation area during the coming 10 years. Moreover, many new mango cultivars,
such as Kent and Tommy Atkins, are being introduced from other countries (e.g.
India and South America) into Egypt.
16
Table 3. Mango imports into European countries (source www.fao.org)
2000 2001 2002 2003

-1 -1 -1
Countries Value t Value t Value t Value t-1
Mt Mt Mt Mt
(103 $) (103 $) (103$) (103$)
U. kingdom 22017 25.98 26957 30.37 24235 26.097 31933 39.555
France 26262 29.96 25693 35.71 26833 30.695 32299 58.404
Netherlands 61856 67.18 69566 68.66 71479 70.907 91133 112.519
Germany 23321 24.93 24825 27.78 27954 29.659 31937 37.09
Belgium 16118 18.07 10292 10.77 10319 11.036 10824 13.597
Portugal 9548 11.3 15189 14.97 15938 14.499 19639 20.696
Switzerland 2794 4.99 3798 6.38 3973 7.372 4808 10.118
Austria 2513 2.46 2438 2.83 2682 3.214 4113 4.224
Spain 9188 10.87 7231 7.64 10410 12.587 11938 15.646
Total 173617 195.74 185989 205.111 193823 206.066 238.624 311.849
Mango problems:
The key features relevant to the profitability of mango production are the
identification of mango market demand and its seasonality prices, seasonal
fluctuations in availability, supply competitors, importer requirements, the option of
added value processing, and sales promotions: the balance between these
components will determine product price (Litz, 1997). To optimize mango
production (harvesting, transferring, shipping, packaging and handling), we need to
focus on production in relation to problems encountered throughout the chain as a
whole. The practical problems relating to mango are conveniently classified into
those which are largely pre-harvest in nature and those that are largely post-
harvest.
Pre-harvest factors:
1. Production seasons are short (2 weeks to 2 months).
2. Varietal appearance and flavour are diverse.
3. Maturation time is variable.
4. Fruit abortion and premature fruit abscission results in yield decreases.
5. Irregular flowering leads to irregular fruiting.
6. There is year to year fluctuation in production
Post-harvest factors:
1. Overall the shelf-life is short.

2. Post-harvest, mangoes ripen rapidly.
17
Chapter 1
3. Mango is sensitive to storage temperatures below 12oC.

4. Anthracnose (Colletotrichum gloeosporoides) is the most important post-
harvest disease, causing huge losses and wastage of mangoes.
The factors that determined the post-harvest behaviour of mangoes are illustrated
in figure 8. They include from farm management during the production phase, the
time of harvest and the availability of the appropriate technology to store and
extend the shelf life of the fruits. The post-harvest performance of the fruits will be
substantially determined by the action of processes that give rise to ripening and
senescence during the post-harvest phase. The activity of these processes is the
result of the interaction of biophysical, biochemical and developmental processes
occurring in the fruits with their physical environment. Thus the appropriate physical
environment can delay ripening and thus increase shelf-life, whereas the incorrect
physical environment will accelerate ripening or cause other forms of product
quality loss, and thus decrease shelf-life. Therefore, understanding the interaction
between the physical environment and product biology is essential if product
storage is optimized. However, not much is known about the biochemical and
biophysical processes of the fruit upon which environmental factors act.
Farm Harvesting Handling

Management Time Fruits
Storage Marketing
Figure 8. Pre and post harvest factors are affected on fruits quality
18
Aim of this thesis
Mangoes, the fruits of the tropical tree Mangifera indica, have a global
production which places them amongst the ten most important fruits in the world.
However in contrast to other major fruits, such as apples, pears or various species
of citrus, the development of appropriate post-harvest technologies for mangoes
has been only poorly studied and now lags a long way behind that of the other
fruits. This results in problems in the storage and transport of mangoes, and thus
less profit and restricted export opportunities for mango producers. Mangoes are a
tropical product and many of the producer countries are classed as
underdeveloped, so increasing the market for mangoes by means of improved
post-harvest management could offer much needed economic opportunities for
these countries.
This thesis will deal with various aspects of the post-harvest physiology of
mango. During the post-harvest phase fruits and other products are subjected to a
range of treatments and conditions as a result of transportation, handling, sorting,
cleaning, storage, packaging, retailing etc. Depending on the perishability of the
product, it will inevitably deteriorate more or less rapidly during the post-harvest
phase as a result of the processes of ripening and senescence. Depending on the
post-harvest conditions and processes this deterioration can be substantially
delayed or accelerated. Thus understanding how postharvest conditions affect the
natural processes of ripening and senescence. A major aim of post-harvest
research is therefore to improve upon our understanding of how this fruit responds
to storage under various conditions so as devise better means of storing mangoes
and measuring the changes in product quality during storage.
The post-harvest phase succeeds production, and though it is often
considered independent of it, this approach is dated and oversimplistic. Current
models of post-harvest quality often attempt to relate post-harvest phenomena with
pre-harvest conditions. This approach recognizes that as the post-harvest phase
succeeds the pre-harvest phase, then practices in the former will have a major
influence on the product performance post-harvest. For any cultivar, post-harvest
behaviour therefore ultimately depends on the pre-harvest growth conditions of the
product, and subsequently how that product responds to the post-harvest treatment
it experiences. To explore these relationships in perennial woody plants (compared
to shorter lived herbaceous crops) is not easy owing to the size of the plants; this
produces difficulties in environmental control, coupled with cost of any treatments
applied and the plants themselves. So the dependency of post-harvest mango fruit
responses upon pre-harvest conditions in addition to the post-harvest environment
is still relatively poorly understood compared to products originating in cool-
19
Chapter 1
temperate or warm-temperate regions. This study will be restricted to investigating

how maturity stage at harvest affects the post-harvest behaviour of mangoes,
especially in relation to their anti-oxidant levels and cell injury developing under low
temperature storage conditions.
Scope of the thesis
Chilling stress during storage leads to typical visible symptoms in various

tissues and disruption of normal physiological function, thereby causing metabolic
imbalance which are often used to quantify and characterize chilling injury
development (Walker et al., 1990). In other words, chilling injured fruits suffer from
physical and physiological changes induced by low temperatures and each
commodity display characteristic symptoms. These symptoms are a function of
plant species, degree of maturity, storage time and storage temperature and other
environmental conditions (Wang, 1990).
Mango fruits are sensitive to low storage temperatures (below 12oC), which
plays a key role in the physiological reactions in fruits that lead to lower metabolic
reaction rates at the chilling temperatures. The most common visible symptoms of
chilling injury in mango fruits are dark skin, scald-like discoloration, and pitting or
sunken lesions on peel. Even abnormal ripening and decay can be enhanced when
mango fruits are exposed to temperatures below 12oC (Chaplin et al., 1991). For
this reason, storage of mango fruits at low temperatures limits safe transporting,
shipping and marketing of mango fruits.
This thesis aims to study the phenomenon of chilling injury in mango fruits
and the various factors affecting its development. Two Egyptian mango cultivars,
Zibdia and Hindi Be-Sennara, were used as models for this research. The former is
chilling sensitive while the other is relatively resistant to chilling conditions. Chilling
determining conditions such as the fruit maturity, storage time and storage
temperature were varied in order to get an insight into the mechanism of chilling
phenomenon in chapter 2. The development of chilling injury symptoms in both
mango cultivars and the variation of chlorophyll fluorescence will be presented in
Chapter 3. Development of chilling injury in fruits is caused by the activities of
antioxidants in fruits. Antioxidants can be classified into water and lipids-soluble
antioxidants such as ascorbic acid, α-tocopherol and β-carotene and antioxidant
enzymes such as superoxide dismutase, catalase, glutathione reductase and
ascorbate peroxidase will be studied in chapter 4 and chapter 5, respectively. The
physiological changes occurring during storage of mangoes such as ethylene
production, respiration rate, fruit firmness, and ion leakage of fruits will be studied in
chapter 6. Thereafter, we aim to present the physiological background of chilling
20
injury phenomena in next parts to aware the input and output metabolic reaction
during long storage.
21
Chapter 1
22
Chapter 2
Chilling injury and the balance between active

oxygen species and the scavenging capacity:
a review
Loay Arafat, Jeremy Harbinson and Olaf van Kooten

Horticultural Production Chains Group, Marijkaweg 22, 6907PG, Wageningen
University, The Netherlands
Chapter 2
Abstract
Chilling injury occurs when certain plants, or organs thereafter, are exposed to
temperatures below 15oC, but above the freezing point of the tissue. After a certain
period of storage at these temperatures, and especially after a subsequent rise in
temperatures, certain injuries in the plant tissue can be seen, such as water
soaking, pitting, or browning. The possible causes of this type of injury will be
reviewed and the consequences for cell components summarized. A distinction is
made between the primary processes of chilling injury and subsequent secondary
injuries. The membrane phase change and oxidative stress models of primary
chilling injury are introduced. How cell components are affected by chilling injury is
then discussed, as is the relationship between pre-harvest conditions, post-harvest
handling, and injury. Lastly, the roles of different anti-oxidants and anti-oxidant
enzymes are discussed and some techniques used for the measurement of chilling
injury are introduced.
24
Review: Chilling Injury and Mango
Chilling injury
Cold storage slows respiration and other metabolic processes that lead to
ripening and senescence of fruits and vegetables during storage. So in order to
extend their shelf life, most fruits and vegetables are cooled as rapidly as possible
after harvest and stored at low temperatures until they are consumed. However, the
shelf life of many fruits and vegetables, especially those of tropical and subtropical
origins (such as mango fruits), as well as some of temperate origin, are actually
shortened when they are stored at low temperatures. For examples banana and
avocado are sensitive to low storage temperatures while green beans, lima beans,
melons or peppers spoil more quickly at low than high temperature (Paull, 1990).
Injury resulting due to exposure to low temperatures above freezing is termed
chilling injury (CI) (Purvis, 2004). CI is an economically important post-harvest
problem that reduces the overall quality and marketability of many harvested fruits
and vegetables indigenous to the tropics and subtropics (Mohammed and
Barthwaite, 2000) Chilling injury may occur in transit or distribution, in retail or
storage (Mitra and Baldwin, 1997). At low storage temperatures, various
physiological and biochemical changes occur in the product’s tissues in response
to chilling temperature. These are categorized as CI. The symptoms of CI vary
depending on the plant species and tissue type and the severity and duration of the
exposure to low temperature. The symptoms usually developed more strongly once
the product is returned to non-chilling temperatures. The extent of chilling injury
damage is hard to quantify and the extent or degree of damage is commonly only
qualitatively or semi-quantitatively estimated using visual inspection of the product,
(e.g. the chilling injury index (Chaplin et al., 1991).
Chilling temperatures induce various structural changes in cell structure and
they disrupt a number of metabolic processes. Consequently several mechanisms
of CI have been proposed. To try and create a rational framework within which
chilling injury could be understood, (Raison and Lyons, 1986) defined CI as the
physical and/or physiological changes that are induced by exposure to low, chilling
temperatures. These physiological changes may be considered primary or
secondary injury. A primary injury is the initial rapid response to low temperature
that causes a dysfunction in plant cell or metabolic process. They considered that
these primary injuries are readily reversible if the temperature is raised to non-
chilling conditions. Secondary injuries arise as a consequence of the primary
injuries, but unlike primary injuries, secondary injuries may be irreversible and thus
they can lead to permanent damage or death whatever the subsequent handling of
the product. A difficulty that has challenged analyses of the origins of chilling injury
25
Chapter 2
is the diversity of processes or mechanisms that have been implicated in its cause,
and the difficulty of reliably distinguishing primary from secondary injuries.
Chilling injury is not the same as freezing injury, which is the result of
damage due to cellular dehydration following extracellular freezing of water and
damage from the ice-crystals that then develop. Freezing damage can only develop
when tissues are held at temperatures below their freezing point. The minimum
safe temperature for chilling sensitive commodities will be substantially above their
freezing point. The critical temperature at which chilling injury symptoms develop
depends upon genetic factors, such as the species and cultivar, and physiological
factors, such as the tissue type, its recent history and its physiological condition.
For a mango fruit the freezing point will be about -1.4oC (Kader, 2002), but storage
temperatures below 12oC are considered unsafe for these fruits (Mitra and Baldwin,
1997). Mangoes, therefore often have a short storage life, as low temperatures
cannot be used to slow deterioration and pathogen growth.
1. Chilling injury symptoms in Mango
Mango fruits are considered to be climacteric* and they ripen rapidly after
harvest. The storage, handling and transport potential of fruits is limited by
susceptibility to diseases, sensitivity to storage temperatures below 12oC, and
perishability due to ripening and softening (Acosta et al., 2000). The combination of
storage temperature and duration of storage are considered to be the important
factors that lead to chilling induced physiological and metabolic dysfunctions in
plant cells. These dysfunctions lead to various visible disorders that are commonly
used to assess the degree of chilling injury experienced by the fruits (Walker et al.,
1990). In mangoes, the most common visual symptoms are dark, scald-like
discolorations in the peel, beginning around lenticels and spreading outwards to
produce a more or less circular lesion, pitting on the fruit peel, the development of
off-flavours, discolouration of the pulp, and overall poor fruit quality (Nair et al.,
2003) (Figure 1).
*
Climacteric: a sudden increase respiratory activity that is correlated with a sudden increase in the
ripening process and a maximum in ethylene production associated with an autocatalytic ethylene
production
26
Figure 1. Severe chilling injury symptoms of Zibdia (a) and Hindi (b) during storage at 4oC for 25
days
2. Chilling injury and its mechanism: the bilayer membrane
The instability of lipid bilayer membranes at low temperatures has long been
thought to be a possible cause of chilling injury. Lipid bilayers are deceptive
structures. Though they seem structurally simple, this belies an underlying
complexity of composition and dynamics. The physiological and structural
properties of the bilayer depend upon its organization, and this depends upon its
composition. Bilayers are comprised of mixtures of numerous types of lipids and
proteins, and not all of the lipids found in cell membranes will naturally form bilayers
in the pure form at physiological temperatures. As temperatures decrease lipid
bilayers will change from their normal flexible liquid-crystalline and functional state
to a more solid gel-like structure. This solidifying of the membranes results in a
decrease in membrane flexibility so cracking can occur and channels can form at
the liquid-crystal/gel interface. These cracks and channels lead to membrane
leakiness, a loss of membrane integrity, and the loss of solute or ion gradients
across the membrane. Further, the transition from the liquid-crystalline to gel phase
produces changes in the stability of the remaining mixture of lipids that comprise
the liquid-crystalline membrane. The gel-phase regions of membrane that form at
low temperatures are crystals that may contain only one lipid species, so the
remaining liquid-crystalline phase will now be enriched in some lipid species and
proteins and deficient in the species crystallised in the gel phase regions. As a
result of the change in lipid composition the remaining liquid-crystal phase will have
different structural and physical properties than the original lipid mixture. The
27
Chapter 2
solidification and phase separation of the membranes also disrupts normal

functioning of membrane bound proteins owing to the change in the lipid
environment of the protein. The reaction rate of these membrane-bound enzyme
systems is inhibited leading to an imbalance in the plant’s metabolism, and the
accumulation of metabolic intermediates which could be toxic to the plant. Leshem,
(1992) has summarized the physiological and biochemical changes due to low
temperatures in plant tissue (Figure 2).
Stress (e.g. Low Temperature)
Time
Membrane Biophysical changes
Temperature
Ethylene
Induce of compositional (chemical)
alternation in membrane lipids
Time (catabolic & anabolic changes)
Temperature
Ethylene
Irreversible later phase separation
(due to accumulation of lipids
degradation production)
Chilling injury symptoms

Figure 2. A model for the sequence of metabolic events that leads from the stress-induced alteration
in the properties of membranes to the observation of macroscopic tissue damage
3. Chilling injury and its mechanism: Oxidative stress
In addition to the role for membrane dysfunction at low temperatures, it is

also becoming widely accepted that the symptoms developing during or after
storage at low temperatures (and also many other environmental stresses) are
actually a consequence of oxidative stress in the tissues (Scandalios, 1993). The
degree of oxidative injury resulting from abiotic and biotic stress imposed on plant
tissues will depend on the one hand upon the oxidative challenge that arises in the
stressed tissue, and on the other upon the cellular capacity to eliminate or deal with
this challenge.
Oxidative stress is due principally to active oxygen species (AOS) derived
from molecular dioxygen. There are various forms of AOS which are formed in
various ways, and the means of formation limits which kind of AOS can be found in
particular tissues under particular circumstances. For example, the formation of
singlet state dioxygen is nearly always dependent on photophysical processes and
28
thus will only occur in light and in the presence of a photophysical sensitiser, such
as chlorophyll. So in contrast to leaves in sunlight, singlet oxygen is unlikely to be a
problem for fruits in dark storage: there is no light and the bulk of the tissue usually
lacks any senstiser. On the other hand, the radical anion superoxide (O2y −) is
formed by the reduction of molecular oxygen (En=-140 mV) (Sun and Trumpower,
2003), and this can occur widely throughout the cell as a parasitic process
associated with formation of reduced cofactors in metabolism.
Most (85% to 95%) (Liu and Huang, 1996; Purvis, 2004) of the oxygen
consumed by non-photosynthesizing or non-photosynthetic plant tissues is reduced
to water by the terminal oxidase(s) of the respiratory electron transport chain in the
mitochondria. The remaining 5% to 15% is partially or fully reduced or incorporated
into organic molecules by various other oxidases and oxygenases. Some electrons
from single-electron-reduced components of electron transfer systems results in the
monovalent reduction of oxygen to the superoxide radical (O2y-). It is estimated that
2% to 4% of the oxygen consumed during normal metabolism is consumed in this
way (Liu and Huang, 1996; Purvis, 2004). In addition to being formed in the
mitochondria, superoxide, can also be formed in different parts of the plant cell,
such as the mitochondria, the chloroplasts, the cell wall and the plasma membrane.
Once formed, superoxide rapidly dismutates (either spontaneously or
enzymatically) to form hydrogen peroxide (H2O2) and molecular oxygen. Hydrogen
peroxide is relatively stable and not a particularly strong oxidant, but it can
penetrate cell membranes relatively easily so it is mobile within the cell. In the
presence of ferrous iron (Fe2+), or other ions capable of a single electron redox
change (e.g. Cu2+), it can be further reduced by superoxide via the Haber-Weiss
reaction to form the highly reactive and detrimental hydroxyl radical (HOy). The
Haber-Weiss reaction is itself just a form of Fenton reaction, a class of reactions
where reductants can facilitate the formation of hydroxyl radicals from peroxides in
the presence of Fe2+ (etc).
3.1. The mitochondria and active oxygen species formation
The mitochondria are major consumers of oxygen, and they are also a major
site of production of AOS (Purvis, 2003). So, for tissues in darkness a major source
of superoxide is the mitochondrial electron transport chain. The respiratory electron
transport chain, located in the inner membrane of the mitochondria of both plant
and animal cells, is made up of five multiprotein complexes, and a mobile
ubiquinone pool which is present in large (10-fold) molar excess compared to the
other components of the electron transport chain. Electron flow from NADH to O2
occurs through three of the protein complexes (complexes III, IV, and I) develops a
29
Chapter 2
proton electrochemical gradient across the inner membrane ( Figure 2). This
electrochemical gradient is dissipated, (i.e., protons flow back across the
mitochondrial inner membrane into the matrix), during the phosphorylation of ADP
to ATP by complex V (the ATP synthase). Thus, a continuous flow of electrons
through the electron transport chain to oxygen requires rapid turnover of ATP to
provide ADP, and this continued flow of electrons to oxygen will serve to re-oxidize
the components of the chain that are reduced by NADH. .
A restriction or disruption of electron flow through the electron transport
chain, such as occurs during resting (state 4) respiration (i.e., when all available
ADP has been phosphorylated to ATP) and in stressed plant tissues, results in a
substantial decrease in oxygen consumption by cytochrome oxidase. As a
consequence, a high concentration of oxygen builds up in the mitochondria and
some of the components of the electron transport chain remain reduced. The high
intra-mitochondrial oxygen concentration and high redox status of the electron
transport chain favour leakage of electrons from reduced components to molecular
oxygen, resulting in increased production of AOS (Skulachev, 1997). Two sites in
the mitochondrial electron transport chain where single electrons can leak to
molecular oxygen reducing it to superoxide are the flavoprotein components of the
dehydrogenases, especially complex I, and the cytochrome bc1 complex (complex
III) (Rich and Bonner, 1978). In addition to complexes I and II, plant mitochondria
have a calcium-activated NADH dehydrogenase located on the exterior surface of
the inner membrane and a rotenone-insensitive NADH dehydrogenase (complex I
activity is inhibited with rotenone) located on the matrix side of the inner membrane
that also reduce the ubiquinone pool ( Figure 2).
The plant uncoupling mitochondrial protein (PUMP) discovered by (Vercesi
et al., 1995) dissipates the proton electrochemical gradient across the
mitochondrial inner membrane without the production of ATP. PUMP is activated by
free fatty acids, especially linoleic acid, but is inhibited by purine nucleotides (e.g.,
GTP). Protonated fatty acids readily cross the mitochondrial inner membrane and
the anionic deprotonated fatty acids are removed by the PUMP from the matrix
back into the inter membrane space resulting in fatty acid recycling and
mitochondrial uncoupling (Jezek et al., 1996). As a result of this uncoupling action,
mitochondrial resting potential (state 4) respiration increases with a parallel
decrease in the trans-membrane electrochemical potential. In addition to
cytochrome oxidase, plant mitochondria have an alternative oxidase (AOX) located
on the matrix side of the inner membrane, that oxidizes ubiquinol directly without
producing a proton electrochemical gradient across the inner membrane. Similar to
cytochrome oxidase, the alternative oxidase reduces oxygen directly to water with
four electrons. Since electron flow through the alternative oxidase is not coupled
30
with the synthesis of ATP, alternative oxidase activity can reduce the redox
potential of the components of the electron transport chain and at the same time
lower the oxygen concentration in the mitochondria (Purvis, 2004; Purvis and
Shewfelt, 1993). Higher alternative oxidase activity requires reduction of the
sulfhydryl groups on the enzyme and the presence of an α-keto acid, such as
pyruvate, the end product of glycolysis (Umbach et al., 1994). Pyruvate interacts
directly with sulfhydryl groups on the enzyme (Umbach et al., 1994), and lowers the
reduced ubiquinone to total ubiquinone ratio required for maximal activity (Day et
al., 1995). Recent studies with transgenic cultured tobacco cells over-expressing
and under-expressing the alternative oxidase show clearly that alternative oxidase
activity lowers the production of AOS when respiratory electron flow is restricted by
cytochrome pathway respiratory inhibitors (Maxwell et al., 1999), and during
phosphate-limited growth (Parsons et al., 1999). Thus, the alternative oxidase
dissipates redox energy without building up the phosphate potential of the
mitochondria.
The ubiquinone molecule can be reduced to ubiquinol by NADH derived
from respiratory substrates oxidized by the TCA cycle enzymes in the mitochondrial
matrix or by other dehydrogenases located in the cytoplasm. Ubiquinol is
subsequently oxidized by complex III (the cytochrome bc1 complex) with the
transfer of electrons one at a time through the cytochrome path to complex IV
(cytochrome oxidase), which ultimately reduces oxygen to water by the
simultaneous transfer of four electrons. A relatively high proportion (almost half) of
the ubiquinone, however, does not appear to be reduced/oxidized by the
31
Chapter 2
Intermembrane space
Succiunate
Fumarate
NADPH
NAD(P)
H+ Complex 2
H+
H+
Cyt
C
Complex 1
QH2
Complex 4
Complex 5
Complex 3
Q QH2 Q QH2
PUMP
Q
Q J2e-
QH2
QH2
QH2
QH2
Q
Q
AOX
H+ ADP
ATP
1/2O2
H2O
NADPH
NAD(P)
1/2O2
H2O
Matrix
H+
Figure 3 .The respiratory electron transport chain of the mitochondrial inner membrane in a plant
cell. The five multi-protein complexes are: complex-1 (rotenone-sensitive NADH dehydrogenase),
complex-2 (succinate dehydrogenase), complex-3 (cytochrome bc1), complex-4 (cytochrome
oxidase), and complex-5 (ATP synthase). The ubiquinone pool is present in large molar excess
relative to the other components. Calcium (Ca2+) activates an external NADH dehydrogenase, a
rotenone-sensitive internal NADH dehydrogenase, a plant uncoupling mitochondrial protein (PUMP)
and an alternative oxidase (AOX). (From Purvis 2004).
respiratory electron transport chain (Wagner and Krab, 1995) and may
participate in the structure of the inner membrane or serve as an antioxidant
(Beyer, 1992).
3.2. Chloroplast physiology as a source of active oxygen species
In chloroplasts AOS are generated from at least three sites in chloroplasts

(Ulrich, 2002). Photosystem I (PSI) can reduce oxygen by Mehler reaction to form
superoxide (see Scheme1) and the rate of this reaction is thought to be increased
32
under conditions where NADP is limiting. Photo-activated, excited chlorophyll

normally transfers its excitation energy to photosynthetic reaction centers where the
energy of the excited state is used to produce charge separation and then electron
transport. However some of this energy, instead of producing photochemistry at the
reaction centre, can excite molecular oxygen from the relatively unreactive triplet
ground state to the much more reactive singlet excited state. Additionally, AOS can
be formed in the photosystem II reaction centre (Telfer et al., 2003) and on the
acceptor side of PSII (Villarejo et al., 2002).
Overall, however, the most important AOS generating reaction in the
chloroplast is the Mehler reaction. The reaction sequence can be illustrated as
follows:
H2O + 2O2 → 2O2- + 2H+ +1/2O2: the overall Mehler reaction
Fd- + O2 → Fd + O2-: reduced ferredoxin is not the only donor
2O2- + 2H+ → H2O2 + O2,: the action of superoxide dismutase
H2O2 → H2O +1/2O2: reduction of peroxide by the ascorbate
Scheme1. The use of oxygen as electron acceptor in a Hill-type reaction by chloroplasts (Mehler,
1951).
Thus the overall electron transfer reaction results in the oxidation of water to
oxygen, electron transport via PSII and PSI which will generate a trans-thylakoid
proton potential, and finally the reduction of oxygen to water (scheme 1). If the
superoxide detoxification reactions of the chloroplast stroma worked perfectly and
destroyed all the superoxide and peroxide produced then there would be no
problem. Inevitably, however, some AOS produces harmful reactions before
destruction.
These reactions are, however, strictly light dependent and cannot occur in
the dark conditions typical of storage. Other sources of AOS in chloroplasts are
associated with metabolism rather than the ‘light reactions’ of photosynthesis
(Robinson et al., 2004) and these could occur in darkness depending on the dark
activation of oxidative metabolic pathways (e.g. the oxidative pentose phosphate
pathway) in the chloroplast stroma and the fluxes of electrons to flavoproteins and
FeS containing proteins. Though metabolic generation of AOS is considered to be
a certainty in the light (Robinson et al 2004), its status in the dark has not been
analysed. Nonetheless, it remains a possibility.
3.3. Cell wall and AOS

Various metabolic reactions which occur in the cell wall are linked to AOS
formation. The most common AOS forming reactions are linked to biosynthetic
33
Chapter 2
reactions, for example, the cross linking of phenylpropanoid precursors by

reactions with H2O2 leading to random linkage of subunits to form lignin (Gross,
1980). NADH is generated via a cell wall malate dehydrogenase, which then form
H2O2 and O2− • as a result of the activity of a bound Mn-peroxidase and other non-
enzymatic reactions (Gross et al., 1977). Diamine oxidase is also involved in the
formation of AOS in the cell wall. This enzyme uses diamines or polyamines
(putrescine, spermidine, cadaverine) as substrates, and it reduces oxygen via a
reduced quinone intermediate to form superoxide (Elstner, 1991; Vianello and
Macri, 1991)
3.4. AOS from the plasma membrane
Superoxide-generating NAD(P)H oxidase activity has been clearly identified in

plasmalemma-enriched fractions (Lesham and Kuiper, 1996). These flavoproteins
may produce superoxide by the redox cycling of certain quinones or nitrogenous
compounds. Toivonen (2004) has reported that low temperature stress causes a
dysfunction of the NAD(P)H oxidase system in plasmalemma which leads to the
formation of superoxide radical (O2•-). Superoxide is also formed in the plasma
membrane of plant cells exposed to fungal elicitors involved in activating the
hypersensitive response found in some plant/elicitor responses (Doke et al., 1991;
Doke and Ohash, 1988). NADH is also possibly oxidized by O2 in the plasmalemma
(Vianello and Macri, 1991).
4. Oxidative stress: sensitivity, tolerance and occurrence
As with any stress, plants normally have two primary strategies of coping with
oxidative stress: they can either avoid it or tolerate it. Fruits and vegetables after
harvest will rarely be in the position to actively avoid stress. Only removing the
commodity from a stress will allow for the avoidance strategy. However, similar to
growing plants, tolerance to oxidative stress of fruits and vegetables in the post-
harvest phase has been associated with factors such as water- and lipid soluble
antioxidants (Hodges and Forney, 2000; Hodges and Forney, 2003), regulation of
AOS production (Duque and Arrabaça, 1999), and membrane composition (Hodges
et al., 2004; Zabrouskov et al., 2002).
34
H2O2 generated from cell wall
H2O2
O2 O2• − J H2O2
Receptor
complex
NADPH
Oxidase Plasmalemma
H2O2
H2O2 O2• −
TCA Cyt.c 2H2O
O2 4e 4H+
Mitochondria
Figure 4.Reactive oxygen species produced by the plant cell at the cell membrane. This figure is
based on information from (Del Río et al., 2002; Mahalingam and Federoff, 2003; Mittler, 2002) and
is also described by (Toivonen, 2004). H2O2 = hydrogen peroxide, O2•⎯ =superoxide anion, TCA =
tricaroxylic acid cycle and Cyti.c = cytochrome c.
Factors determining sensitivity to, or occurrence of, oxidative stress
In addition to internal factors, such as antioxidant pool sizes, the physical

environment of the product and its handling have a major influence on the
development of oxidative stress. Below is a summary of the factors that are
considered important in determining the degree of oxidative stress or injury that
develops in a product post-harvest.
1. Fruit or vegetable cultivar (i.e. genotype) and harvest maturity (Gong et al.,
2001; Masia, 1998).
2. Harvest procedures and storage duration (Hodges and Forney, 2000).
3. Storage temperature (Wismer, 2003).
4. Storage atmosphere (Gunes et al., 2002).
5. Processing protocols (Hodges et al., 2000; Pirker et al., 2002).
6. Conditions exacerbating water loss (Toivonen, 2003).
7. Senescence and/or ripening of the commodity (Aharoni et al., 2002; Lacan
and Baccou, 1998; Lester, 2003).
35
Chapter 2
4.1. Harvest maturity
Oxidative injury disorders are highly dependent upon the harvest maturity stages of
the fruit or vegetable, an observation hypothesized to be related to scavenging
capacities of water and lipid-soluble antioxidants such as ascorbate, or tocopherols
and carotenoids, and the antioxidant enzymes superoxide dismutase (SOD),
catalase (CAT), peroxidase (POX) , ascorbate peroxidase (APX), and glutathione
reductase (GR) (Toivonen, 2003). For example, disorders such as fruit scald have
been associated with decreased antioxidant capacity (DeLong and Prange, 2003;
Larrigaudiere et al., 2001). And are related to harvest maturity (Toivonen, 2003).
Kader (2002) reported that the concentration of water-soluble antioxidants (such as
ascorbic acid) in mango fruits depends on their maturity stage at time of harvest. It
is higher in immature fruits (M1) compared with half (M2) and fully mature (M3)
fruits at harvest time whereas the reverse trend was observed for the carotenoids.
Barden and Bramlage, (1994) considered that the levels of lipid-soluble
antioxidants during apple storage depended on maturity stage, and that the higher
antioxidant content that developed at full maturity was necessary to avoid scald.
Maturation of Saskatoon fruit (Amelanchier alnifolia Nutt.) is accompanied by
increases in oxidative stress and a parallel decline in SOD and CAT activities. The
anthocyanin levels in blueberry fruits increased with harvest maturity, though both
the oxygen-radical absorbing capacity and phenolic content were lower in more ripe
fruit (Kalt et al., 2003). These results are apparently cultivar and/or growing region
dependent as another study with the blueberry cultivar ‘Elliot’ showed increases in
antioxidant activity, total phenolic content and anthocyanin content with fruit
maturity (Connor et al., 2002). Tomatoes picked at the fully ripe stage also
developed higher levels of lycopene, β-carotene, and ascorbate than fruit picked
stages where they are unripe (Giovanelli et al., 1999). From this it can be
concluded that changes in total antioxidant content or activity of a fruit or other
product during ripening, storage etc cannot be known if the changes of only a
limited number of anti-oxidants are measured.
4.2. Storage temperature
Control of storage temperature is used to optimise or maintain quality

attributes of fruits and vegetables, such as texture, flavour and appearance, over
time. Low-temperature storage extends the shelf life of plant commodities, but
product-specific temperature thresholds exist beyond which symptoms of chilling-
induced oxidative injury and oxidation-related disorders will occur (Hodges et al.,
2004; Toivonen, 2003; Wismer, 2003). Even the recommended optimal storage
temperatures may accelerate oxidative stress and induce senescence (Zhuang et
36
al., 1997). The induction of oxidative stress by chilling temperatures has been well
studied. In broad terms, in chilling-sensitive commodities, chilling temperatures
impair the energy state of the cell, disorder metabolism, and/or provoke alterations
in membrane integrity (Bartosz, 1997; Purvis, 2003; Purvis and Shewfelt, 1993).
This often results in increased AOS formation, while also reducing scavenging
efficacy through such factors as chilling-related inactivation of antioxidants and/or
impeded antioxidant turnover (Bartosz, 1997; Wismer, 2003). Membrane lipid
peroxidation may be one of the first events in the development of chilling injury
(Tijskens et al, 1994). Profiles of antioxidant compounds may also be altered in
response to low temperature extremes.
Sala and Lafuente (2000) found that SOD activity of the flavedo (the
pigmented skin) of mandarin fruits increased in both chilling-sensitive and chilling-
tolerant varieties, while CAT, ascorbate peroxidase (APX), and GR increased only
in the chilling-tolerant cultivars. Purvis et al (1995) proposed that mitochondria are
a source of oxidative stress during chilling as superoxide production from the
mitochondrial electron transport chain is increased in chilling-sensitive green bell-
peppers. A wide range of storage disorders, such as low-temperature sweetening,
superficial scald, pitting, and core browning, are related to low-temperature-induced
oxidative stress (Toivonen, 2003; Wismer, 2003).
An effect of chilling temperatures is to alter the homeostasis between AOS
generation and defence mechanisms with the result that the net AOS burden
increases and damage ensues (Hodges, 2003). The working hypothesis that arises
from this simple model is that chilling-tolerant plants or plant parts may initially
contain more, or can generate more, antioxidants during stress and/or produce
fewer AOS than more chilling-sensitive plant species (Wismer, 2003).
4.3. Water status
Hodges et al (2004) reported that there was a relationship between water

loss during the post-harvest storage and AOS generation, and high humidity
during storage has been shown to reduce chilling injury (Forney and Lipton, 1990).
Moreover under non-chilling conditions, wilting has been associated with
decreases in antioxidants, such as ascorbic acid, in cabbage and snap bean
(Toivonen, 2003). Water loss has been shown to cause an increase in the
production of AOS, especially H2O2, in plant tissues (Yan et al., 2003). For
example, in muskmelon (Lester and Bruton, 1986) and mango fruits (Pesis et al.,
2000) it has been found that a high humidity achieved by MAP or a plastic film
package with a selective gas permeability prevented the development of chilling
injury and prevented the loss of carotene, fruit sugars, non-enzymatic browning,
37
Chapter 2
membrane integrity and fresh weight, while maintaining fruits firmness and
marketable appearance. In retail displays, using water mists at non-refrigerated
temperatures reduces both tissue water loss and promotes the retention of ascorbic
acid and total carotene in muskmelon fruits (Barth and Zhuang, 1996).
4.4. Processing
Physical abrasions, high or low processing temperatures, and product

preservation techniques may all induce an increase oxidative stress (Hodges et al.,
2000). For example, Pirker et al. (2002) stated that the AOS content of freeze-dried
strawberry samples was approximately 10 times higher than that of frozen
strawberries samples. In a study comparing air and MAP storage, followed by
cooking in water, on the ascorbate content of fresh-cut spinach, a decrease in the
total antioxidant activity was observed during 7 days of storage, but with ascorbate
levels remaining higher in MAP atmospheres (Gil et al., 1999). Subsequent boiling
of the spinach in water for 10 min resulted in a 60% loss of ascorbate to the
surrounding water.
Irradiation with ionizing radiation, such as gamma rays or electron beams,
induces the development of AOS that can lead to oxidative disorders in both fruit
and vegetable tissues (Toivonen, 2003). Ionizing irradiation produces ozone both in
air and any in oxygen-containing plant tissues, especially in air-filled apoplastic
spaces (Maxie and Kader, 1966). These increases in AOS are in addition to the
primary damage that ionizing radiation causes to plant membranes, proteins and
DNA (Jones and Bulford, 1990).
Various disorders due to post-harvest irradiation of products have been
reported by Hodges et al (2004) and many involve decreases in anti-oxidative
capacity. Reported problems include: lower sensitivity to ethylene in apples
(Abeles, 1973), increased electrolyte leakage (Lester and Wolfenbarger, 1990) and
decreases in the ascorbic acid (Garcia-Yanez et al., 1990.) and glutathione
(Toyo'oka et al., 1989) levels in grapefruit, de-esterification of phospholipids in
cauliflower (Voisine et al., 1991), and losses of carotenoids in potatoes (Mitchell et
al., 1990). Lester and Wolfenbarger (1990) found that a 0.25 kGy dose greatly
increased measurable membrane damage (electrolyte leakage), loss of total
phenols and ascorbic acid levels in fresh grapefruit. However although there are
many examples of oxidative disorders developing due to ionizing irradiation, the
decline in marketable quality and nutritional value may be contained within
acceptable limits if appropriate doses/rates are applied (Toivonen, 2003). (Kader,
1986) has summarized the recommended safe doses of ionizing irradiation for
fresh fruits and vegetables.
38
Ultra-violet irradiation in the UV-B and UV-C bands can also can affect AOS
levels in plant tissues. Schmitz-Eiberger and Noga (2001) found significant
changes in green bean seedling leaves following UV-B (280-320 nm) irradiation.
UV-B-induced injury resulted in a disturbance of the active oxygen metabolism
system by destroying non-enzymatic antioxidant mechanisms ( for example the
ASC pool) and altering the enzymatic antioxidant defence systems ( for example
SOD and APX activity decreased). Barka (2001) showed that immediately upon
exposing tomato fruit to a low dose of UV-C irradiation (3.7 kJ·m–2 at 254 nm),
superoxide dismutase and ascorbate oxidase activities were decreased while those
of lipoxygenase and phenylalanine ammonia lyase were increased. Much is still
unknown about the development of oxidative stress in fresh fruits and vegetables
in relation to energetic irradiation of various kinds, and this is, therefore, still an
important area for research (Hodges et al., 2004).
4.6. Ripening and AOS
Ripening is associated with rapid changes in cellular components, an increase in

catabolic processes, and the initiation of senescence (Masia, 2003). So, ripening, which
involves developmental changes in color, firmness, sugar, flavor, and proteins, can be
differentiated from senescence as the latter involves a loss of membrane integrity and
tissue death (Noodén, 1988). During ripening, levels of AOS progressively increase,
due mainly to the declining activity of antioxidant enzymes that diminish AOS levels.
Aharoni et al. (2002) conducted a comprehensive investigation of gene expression
during the maturation and non-climacteric ripening of strawberry. Gene expression of
ripening fruits was compared to gene expression in the fruits subjected to oxidative
stress following their treatment with the free-radical generator 2,2’-azobis(2-
propanimidamide) dihydrochloride. It was found that of the 46 genes that were induced
by oxidative stress, 20 genes were also upregulated during ripening, and of these some
were clearly linked to anti-oxidative activity (e.g. ferritin, glutaredoxin, glutathione-S-
transferase)
Rogiers et al (1998) clearly showed that the ripening of climacteric
Saskatoon fruit (Amelanchier alnifolia Nutt.) is associated with a rise in AOS that
affects membrane lipids. The increase in lipid peroxidation during fruit ripening was
suggested by the evolution of ethane and the development of thiobarbituric acid-
reactive substances (TBARS), both of which are indicators of the breakdown of
oxidised membrane lipids. At the same time the activities of SOD and CAT
enzymes declined 4-fold and 18-fold, respectively, and under these circumstances
cytotoxic levels of H2O2 might accumulate. Lipoxygenase activity increased 2.5-fold
during ripening, while the reduced and oxidized forms of glutathione increased
39
Chapter 2
during the later stages of ripening with only a minimum (3%) variation of the
percentage of GSSG relative to the total of (GSG+GSSG). In ripening pepper fruits,
higher ascorbate peroxidase and Mn-SOD activities in the mitochondria play a role
in avoiding the accumulation of AOS (Jiménez et al., 2003). The presence of
elevated levels of the antioxidant enzyme system implies that the management of
oxidative stress is a key factor during fruit and vegetable ripening.
4.5. Senescence
Senescence is considered the terminal phase of life of plant organs,

including leaves, flowers and fruits (Buchanan-Wollaston, 1997). The most
intensively investigated plant tissue regarding senescence and AOS are those of
leaves. In senescing leaf systems lipid peroxidation is typically associated with a
decline in catalase and superoxide dismutase activities, increased levels of H2O2
and reduced transcription of mRNA (Watkins and Rao, 2003). Few studies have
attempted to identify differences in AOS activities in relation to different rates of
senescence among fruits and vegetables (Hodges et al., 2001). (Du and Bramlage,
1994) found that total SOD activity (i.e., CuZn-SOD, Fe-SOD and Mn-SOD)
changed greatly during senescence of various apple cultivars, while (Jiménez et al.,
2003), comparing green versus red peppers (Capsicum annuum), found that
numerous antioxidants play a role in pepper senescence. Superoxide diumutase
isozymes (Mn-SOD, Fe-SOD and CuZn-SOD), GR, CAT and APX exhibited higher
activity levels in the red peppers, which were more senescent, than in the green
fruits, which were less senescent. In contrast, however, monodehydroascorbate
reductase and dehydroascorbate reductase activities were higher in the green fruit.
These correlations imply that SODs, CAT and APX are probably involved in pepper
fruit senescence.
4.7. Ethylene
The processes of ripening and senescence in fruits and vegetables are

associated with oxidative mechanisms (Masia, 2003). Ethylene is commonly
considered as the hormone that regulates ripening, and exposure of sensitive plant
tissues to this phytohormone results in acceleration of both ripening and/or
senescence (Hodges and Forney, 2000). However, oxidative processes, such as
lipid peroxidation, which are induced in some fruits in response to ethylene
exposure (Meir et al., 1991), appear to be secondary effects and not as a direct
result of ethylene exposure (Toivenen, 2003). In addition, many commodities are
insensitive to ethylene and in these cases the processes of ripening and
senescence are not accelerated by exposure to the hormone (Palou et al., 2003).
40
Ethylene synthesis, on the other hand, is enhanced by environmental and biological

stresses such as wounding or pathogen attack (Morgan and Drew, 1997; Ohtsubo
et al., 1999) and it may be stimulated by AOS and lipoxygenase activity during
membrane lipid peroxidation, further accelerating ripening and senescence
processes (Hodges, 2003).
5. Oxidative reactions in stressed tissues
Oxidative stress occurs when the generation of active oxygen species (AOS)
exceeds the capacity of the plant to maintain cellular redox homeostasis, or, more
simply, when the production of AOS exceeds the capacity of the plant to scavenge
them. Lipids, proteins, carbohydrates, and nucleic acids are all targets of AOS
(Hodges, 2003). Chloroplasts, mitochondria, nuclei, glyoxysomes, and peroxisomes
all represent major sites of AOS production, though the importance of
chloroplastidic sources is likely to decrease in ripening fruits and commodities
which are usually stored in dark conditions. Membranes contain unsaturated fatty
acid residues which are particularly vulnerable to oxidation, so oxidative damage to
membranes is of particular importance.
5.1. Lipid peroxidation
The lipid bilayer membrane is composed of a mixture of phospholipids and

glycolipids in which the fatty acid chains are attached to the C1 and C2 of the
glycerol backbone by ester linkages. The peroxidation reactions are different
among different fatty acids and depend upon the number and position of double
bonds in the acyl chain. Lipid peroxidation reactions involve the three distinct steps
typical of free-radical chain reactions: initiation, propagation, and termination
(Franke, 1985). In the initiation step an AOS, such as a hydroxyl radical, reacts with
an unsaturated fatty acid residue (e.g. Linoleate) in the hydrophobic core of the
membrane abstracting an H atom and forming a methylvinyl radical on the fatty
acid chain:
OHy +RH Æ R• + H2O
This and subsequent reactions are also illustrated in figure 4; in this case a
hydroxy radical reacts with carbon number 11 of the linoleate residue to form a
radical which is partly stabilized by being delocalized across the two conjugated
double bonds that now exist between carbon atoms 9 to 13 (Figure 5). During the
propagation reaction, this free-radical reacts with normal ground state triplet
oxygen, which is a biradical, to form a peroxy radical:
R• + O2 ÆROO•.
41
Chapter 2
In the case of linoleate when this reaction occurs on carbon atoms 9 or 13

the peroxy radical formed abstracts a hydrogen atom from another fatty acid
molecule to form a hydroperoxide and creating another vinyl radical on the attacked
lipid. This can then react with oxygen (as before), or abstract a hydrogen from
another lipid which though eliminating the first radical, creates another radical
which can react with oxygen etc:
ROO• + RH Æ R• + ROOH
Degradation
Products
D
Figure 5. The peroxidation of linoleic acid. The hydroxyl radical abstracts an H atom from carbon-11
of the fatty acid between the two double bonds and is thus converted to water. The free radical that
is produced on the fatty acid is delocalized between carbons 9 to 13 and is thus to some degree
stabilised. Triplet molecular oxygen (the ground state that has two unpaired electrons and may
attack this structure at either carbon -9 or -13 forming a alkyl-peroxy radical. This peroxy radical can
then react in various ways, for example by abstracting another hydrogen atom from a second
linoleic acid molecule in a propagation reaction forming a lipid hydroperoxide as shown in the
diagram. The various peroxo and other oxo-adducts that are formed are unstable and various
bearkdown reactions, such as chain breakage and cross-linkage reactions subsequently occur to
produce aldehydes, hydrocarbons, alcohols, and cross-linked dimers.
The role of initial hydroxy radical is to start an oxidative reaction chain

reaction that forms unstable, reactive lipid peroxides and free-radicals. The
peroxidised lipid chains disrupt membrane structure owing to their hydrophilic
character and they may spontaneously break down forming a wide range of
products, some of which are toxic or reactive. Typical lipid peroxidation degradation
products of ROOH are aldehydes, such as malondialdehyde, and hydrocarbons,
such as ethane and ethylene. The termination reaction, which stops the free-radical
chain reaction, can be a reaction between two lipid free-radicals, or a reaction
between a free-radical and a membrane anti-oxidant such as tocopherol (Baier and
Dietz, 1999)
42
5.2. Protein oxidation
During the oxidation of proteins, oxidation starts on the hydrocarbon portion

of protein molecule in the vicinity of amino acid group, and this leads to amino acid
modification, followed possibly by fragmentation of the peptide chain. Protein
oxidation reactions are initiated by the attack of •OH and abstraction of α-H atom of
amino acid to form a carbon-centred radical. This can abstract a hydrogen from a
nearby group thus forming a new radical, which may react and cross-link with an
adjacent organic radical, or it may react with molecular oxygen to form an
alkylperoxyl radical. This radical can be reduced to an alkylperoxide by superoxide
in the presence of a ferrous ion (or similar) catalyst and the peroxide formed may
then be cleaved to form an alkoxyl radical again by superoxide in the presence of a
ferrous ion catalyst. Finally, the alkoxyl radical can be reduced to an alcohol by
reduction by superoxide in the presence of a ferrous ion catalyst. Though reduction
to an alcohol is one possibility, other reactions can occur that result in peptide chain
crosslinkage, cleavage, or reactions with other molecules, such as sugars or lipids.
One of the most important products of the oxidative reactions are the protein
carbonyl derivatives. These are produced by a range of reactions, for example by
the cleavage of peptide chain by the α-amidation pathway or by the oxidation of
glutamyl side chain leading to the formation of NH3 and free α-ketocarboxylic acid.
(Berlett and Stadtman, 1997). Oxidation of lysine, arginine, proline, and threonine,
will also yield carbonyl derivatives. In addition, carbonyl groups may be introduced
into the peptide chain by reactions with the beakdown products of peroxidized
lipids, such as the aldehydes 4-Hydroxy-2-nonenal and malondialdehyde
(Schuenstein and Esterbauer, 1979; Uchida and Stadtman, 1993). The presence of
carbonyl groups in proteins has been widely used an indicator of AOS linked
oxidation of cell constituents, though the diverse routes by which these carbonyl
groups are formed can complicate simple interpretation of the results. Several
sensitive methods for the detection and quantition of protein carbonyl group have
been developed (Levine et al., 1994).
6. Postharvest oxidative disorders
The classical post-harvest disorders associated with oxidative damage

disorder, as documented by Purvis (2004), include superficial scald (DeLong and
Prange, 2003) and core browning (Larrigaudiere et al., 2001), bleaching of
pigments (Elstner and Osswald, 1994) and disruptions in membrane integrity
(Biedinger et al., 1990). The inactivation of many types of functional proteins (e.g.,
enzymes) resulting from damage is followed by increased protease activity
43
Chapter 2
(Casano et al., 1994; Landry and Pell, 1993), and lesions and damage to nucleic
acids are also common postharvest- disorders associated with oxidative stress.
Active oxygen species have also been implicated in the regulation, properties,
and/or dynamics of induced or natural senescent processes (Droillard et al., 1987;
Hodges and Forney, 2003; Philosoph-Hadas et al., 1994; Thompson et al., 1991).
A major characteristic of senescence in plant tissues is increased lipid peroxidation
(Kunert and Ederer, 1985; Lacan and Baccou, 1998). Lipid peroxidation is thought
to play an important role in the biosynthesis of ethylene (Paulin et al., 1986), a
hormone involved in the regulation of senescence.. Although the exact mechanism
of ethylene synthesis under these circumstances is unclear, but it may arise as a
lipid-fragmentation product through following the oxidation of unsaturated centres in
the acyl chains of fatty acids by lipoxygenase (Gardner and Newton, 1987). This
enzyme catalyzes the production of hydroperoxy conjugated dienes from
polyunsaturated fatty acids.
7. Defence mechanism against AOS
In addition to causing damage to a range of cell components, AOS also

serve as signals or messengers that trigger an increase in the production of
antioxidants and active oxygen scavenging enzymes (Vranová et al., 2002). They
are also involved in programmed cell death, which they can modulate in two ways.
They can act as signalling molecules to initiate cell death or they can directly kill the
plant cell (Fath et al., 2002). They are also involved with the senescence of plant
tissues (Rubinstein, 2000). The level of AOS in plant tissues must therefore be
regulated. This regulation is achieved by a variety of mechanisms, which includes
an elaborate scavenging system that depends upon active oxygen scavenging
enzymes such as superoxide dismutase (SOD), catalase (CAT) and the various
peroxidases, and a range of lipid-soluble (α-tocopherol and the carotenoids) and
water-soluble (ascorbic acid, glutathione and the flavonoids) antioxidants (Noctor
and Foyer, 1998).
7.1. Water-soluble antioxidants.
Ascorbic acid is an important quality component of many fruits. It is also an

essential constituent of the human diet. Apart from preventing the well-known
vitamin C deficiency disease scurvy, this substance has gained increasing
recognition as playing a diverse role in maintaining human health. It is now
generally accepted that vitamin C is one of the most important free radical
scavengers in plants, animals and humans (MacKersie and Lesham, 1994). In
44
plants, ASC is found in the chloroplast, cytosol, vacuole and extra-cellular

compartments (Foyer and Halliwell, 1976). Ascorbic 1acid can directly and
indirectly scavenge AOS (Figure 6) with or without the involvement of enzyme
catalysts, and indirectly by recycling oxidized tocopherol to the reduced form
(Foyer, 1993). It reacts with superoxide, hydrogen peroxide or the tocopheroxyl
radical to form monodehydroascorbate and/or didehydroascorbate. These oxidised
forms can be recycled back to ascorbic acid by means of monodehydroascorbate
reductase and dehydroascorbate reductase using reducing equivalents from
NADPH and glutathione, or the dehydroascorbate may decompose into tartaric and
oxalic acid (Figure 7 and 8) (Foyer 1993). Furthermore, ASC is thought to protect
α-tocopherol (α-TOC) and recycle α-tocopheroxyl radical as shown in figure 9
(Munné-Bosch and Alegre, 2002; Smirnoff and Wheeler, 2000).
Figure 6. Oxidation of ascorbic acid to dehydroascorbic acid, modified from (Bors and Buettner,
1997; Niki, 1991)
Figure 7. Halliwell-Asada pathway or ascorbate-glutathione cycle: APX, ascorbate-peroxidase;

MDHAR, onodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione
reductase. From (May et al., 1998).
45
Chapter 2
Figure 8. The degradation of L-ascorbic acid in plant tissues
7.2. Lipid-soluble antioxidants

7.2.1. α-Tocopherol (α-TOC)
α-Tocopherol acts to chemically stabilize the inner regions of the membrane

bilayer. It achieves this feat owing to its properties as an antioxidant which can
chemically and physically scavenge a range of AOS, such as oxygen free radicals,
lipid peroxyl radicals and singlet oxygen (Diplock et al., 1989). α-Tocopherol
synthesis occurs in plastids, including chloroplasts (Bramley et al., 2000). The
aromatic ring is formed by the shikimic acid pathway and the phytyl chain is
synthesized from the geranylgeranyl pyrophosphate and terpenoid pathways. The
antioxidant properties of α-tocopherol are based upon its ability to quench both
singlet oxygen and peroxide, though it is a less efficient scavenger for 1O2 than β-
carotene. It is also able to react with the peroxyl radical formed by the addition of
O2 to the vinyl radical formed on the fatty-acid tails of membrane lipids. This
reaction terminates the radical chain reaction because even though a tocopheroxyl
radical is the product of the reaction, this radical is stable and does not react
further. As a result, tocopherol is considered an effective free radical trap. An
important feature of the tocopheroxyl radical is that the radical is stabilised by being
46
delocalised on the phenol ring (Figure 9), which, owing to its hydroxyl group, is also
the most hydrophilic part of the molecule. It is this part that will be near the surface
of the bilayer, which is also where the hydrophilic peroxyl radicals will be found and
where the tocopheroxyl radical can be most easily reduced by water-soluble
ascorbate (Foyer, 1992).
Figure 9. The formation of the α-tocopheroxyl free radical and its subsequent reduction to
tocopherol: (a) Initiation of the peroxidation reaction when a free-radical X•, abstracts a hydrogen
atom from an unsaturated acyl chain forming a linear pentadienyl radical.(b) this is then oxygenated
to form a peroxyl radical and a conjugated diene. (c) The hydrophilic peroxyl radical moiety moves
to the water-membrane interface where it available for repair by tocopherol. (d) the peroxyl radical is
reduced to a lipid hydroperoxide, and the tocopherol oxidized to a tocopheroxyl radical, which can
then be reduced by ascorbate. (e) Tocopherol has been recycled by ascorbate; the resulting
ascorbate radical can be recycled by enzyme systems. A range of enzymes including phospholipase
A2 (PLA2), phospholipid hydroperoxide glutathione peroxidase (PH-GPx), glutathione peroxidase
(GPx) and fatty acylcoenzyme A (FA-CoA) may then reduced and repair the oxidized acyl chain of
the phospholipid (Buettner and Schafer, 1999).
47
Chapter 2
7.2.2. β-Carotene (CAR)
Carotenoids are C40 isoprenoids and tetraterpenes that are located in

plastids of photosynthetic and non-photosynthetic plant tissues. In chloroplasts, one
function of carotenoids is to act as an accessory pigment in light harvesting.
Another important role in chloroplasts is the to detoxify AOS and convert triplet
chlorophyll produced during photosynthetic light-harvesting to the singlet ground
state Triplet chlorophyll is not harmful, but it is potentially relatively long-lived and
thus it could react with triplet ground state molecular oxygen to form the highly
reactive singlet excited state (Hideg et al., 1998). 3Chl* can transfer its excitation
energy and triplet character to adjacent carotenoid molecules to form triplet
carotenoids and singlet ground state chlorophyll. The triplet carotene then decays
very rapidly to the ground state, thus eliminating the triplet character of the
molecule and preventing singlet oxygen formation:
3
Chl* + 1β-car J 1Chl + 3β-car*
3
β-car* J 1β-car + heat
Another carotenoid, zeathanthin plays a particularly important role in the
regulation of photosystem II. This xanthophylls acts in a way that it is not yet fully
understood to facilitate the de-excitation of excited singlet chlorophyll to the ground
state, with the release of the excitation energy as heat. This quenching mechanism
competes with the reaction centres of photosystem II as quenching route, and thus
reduces the likelihood that charge separation will occur in the reaction centre. This
results in a reduction in the quantum efficiency of electron transport by
photosystem II. Under light-limiting conditions this loss of efficiency would result in
a loss of CO2 fixation capacity, but at higher light intensities this loss of quantum
efficiency seems to act to protect photosystem II reaction centres from
photoinhibition, a process which also seems to involve active oxygen.
7.3. Antioxidant enzymes
The continuous oxidative stress that cells are under results in their having a
comprehensive protection system to guard against injury from AOS. The extent to
which this protective system increase its activity in response to an oxidative
challenge, or the extent to which the increase in oxidative damage correlates with
decreases in the activity of certain protective mechanisms is valuable as a priori
evidence of possible regulation of control systems and causal pathways leading to
48
injury. Plants have both enzymatic and non-enzymatic antioxidant systems to

prevent or alleviate the damage from AOS. Many enzymes can efficiently reverse
or detoxify the effects of AOS (e.g. lipid hydroperoxidases (Baier and Dietz, 1999),
whereas others act to detoxify various AOS before they can cause damage, for
example O2•- is detoxified by SOD, and H2O2 is removed by CAT and by reactions
catalysed by different kinds of peroxidases (e.g. guaiacol peroxidases (GPX). A
major H2O2 detoxifying system in plants is the ascorbate-glutathione cycle that
includes APX and GR (Asada, 1994). These protection systems are not perfect,
however, and an increase in the production of AOS or a decrease in scavenging
capacity will result in an increase in oxidative damage. This will first produce an
increase in lipid and protein oxidation followed by the development of classical,
oxidative postharvest disorders (Hodges et al., 2004).
Generally, however, plants show a rapid response to increased AOS produced
by physical stresses (in response to, for example, changes in storage conditions
e.g. temperature and time (Rogiers et al., 1998). These changes lead to an
enhancement in cellular oxidants that induce an increase in the activities of non-
enzymatic antioxidants such as ASC, α-TOC and β-CAR, and antioxidant enzymes
activity such as SOD CAT, GR, and APX (Bowler et al., 1992; Foyer and
Harbinson, 1994). The effects of storage conditions on the actions of the natural
antioxidants are studied in details in chapter 4 and 5. (Saltveit and Morris, 1990)
have reported that many fruits have differing sensitivities towards chilling
temperatures depending on their maturity stage or stage of development once fully
mature. Studies on the chilling sensitivity of pre and post-climacteric fruits have
shown that pre-climacteric Avocado, papaya and mango fruits are more sensitive
to low storage temperatures (Robert, 1990).
8. Increasing chilling tolerance chilling injury
Chilling sensitivity is observed in many plants or parts of plants whose vitality or

post-harvest longevity are diminished by exposing the them to temperatures from
0oC (non-freezing) to about 15oC (Wang, 1990). A range of processes have been
suggested to be the causal factor differentiating chilling tolerant from chilling
sensitive tissues, cultivars or species. There are also many different studies
investigating the genetic basis for this physiological phenomenon and exploring its
variability. Genetically, variability for chilling tolerance exists in most crop species,
such as tomato (Walker et al., 1990) and peanut (Bell, 1993). Many breeding
programs have attempted to transfer the genes of chilling tolerance from tolerant
plants into sensitive plants, but this is often a complex and time consuming
process. Chilling tolerance is usually found in distant wild plant relatives of
49
Chapter 2
commercial plants. When those plants were used in plant breeding programs to
increase the tolerance of the sensitive plant there was also loss of commercial
quality. Breeding for improved or heightened oxidative stress defense in cucumber
fruits by increasing the CuZn-SOD levels was recently accomplished by transgenic
manipulation (Lee et al., 2003).
An alternative approach is to exploit the cross-tolerance phenomenon were
increasing a plant’s resistant to one stress increases its resistance to others. For
example heat stress will provoke physiological responses that also increases the
chilling resistance. Heat treatment of mangoes in air or water will reduce chilling
injury symptoms and increase their shelf-life, but these treatments have a negative
effect on mango quality and are therefore unusable in practice (Majeed and Jeffery,
2002).
9. Methods for the assessment chilling injury
In addition to assessing the degree of chilling injury by means of scoring the

visual symptoms of injury (Walker et al., 1990) a range of other techniques are
available with which to assess damage.
9.1. Chlorophyll fluorescence
One of the most sensitive responses to low storage temperatures is damage

to photosystem II. This may be measured by means of the non-destructive
chlorophyll fluorescence (CF) technique. It is important to note that damage to
photosystem II can be produced not only by exposure of chilled tissue to a
relatively high irradiance, but also by storing tissue in darkness under chilling
conditions. The damage to photosystem II provoked by the combination of light and
chilling is a well studied phenomena called photoinhibition, and it is probably a
consequence of photochemical activity in the photosystem II reaction centre
resulting in singlet oxygen formation rather than electron transport. Damage to
photosystem II provoked by low temperatures in darkness develops under more
severe conditions (lower temperatures, longer duration of chilling) and its
physiological basis is not known, though clearly it cannot involve photochemically
induced injury as light is absent. Phase transitions in the thylakoid membrane have
been associated with the dark-induced damage to photosystem II activity
temperature that coincide with temperature of the phase transition in the polar lipids
of thylakoid membrane (Meir et al., 1995; Parkin et al., 1989). Damage to
photosystem II, measured by means of chlorophyll fluorescence, is both a very
sensitive and convenient indicator of damage to the thylakoid membrane, as shown
in apple (DeEll, 1996) and cucumber (Tijskens et al., 1994). The chlorophyll
50
fluorescence parameter used to assess damage to photosystem II is the Fv/Fm

ratio, Fm is the maximum relative fluorescence yield produced in dark-adapted
material subjected to an irradiance sufficient to reduce all the QA pool, Fv is Fm
minus F0, where F0 is relative fluorescence yield measured from dark-adapted
material when the QA pool is oxidised (i.e. non-reduced). An Fv/Fm ratio near 0.8 is
typical of healthy, unstressed material (Björkman and Demmig, 1987; Bolhàr-
Nordenkampf et al., 1989). Reductions in Fv/Fm below this value are widely
considered as being indicative that the material has been stressed, though often
the causal relationship between the stress and the reduction in Fv/Fm is not
explored or explained.
Because of its convenience, the CF technique has been explored as a
possible tool for the quantitative assessment of post-harvest chilling injury
produced during storage: it is rapid, sensitive, non-destructive to tissues and able to
detect injury before visible symptoms appear (DeEll, 1996; MacRae et al., 1986;
Schreiber and Bilger, 1987). Different applications of CF in the post-harvest phase
during storage under low temperatures have been used to evaluate chilling injury
development in many fruits; for example banana (Musa acuminata Colla) and
mango (Mangifera indica L.) (Smillie et al., 1987), in cucumber (van Kooten et al.,
1992) and green pepper (Capsicum annuum L.) (Lurie et al., 1994).
9.2. Ion leakage
Ion leakage is a widely used technique for measuring damage to tissues,

especially following low temperatures stresses, such as chilling or freezing, applied
to sensitive plants. Ion leakage requires that the cell membrane looses its
semipermeable nature, but whether this is a primary or secondary effect of the
stress depends on the mode of action of the stress. The two syndromes that could
lead to leakage as a result of chilling injury are summarized below (Hariyadi and
Parkin, 1991), in one case injury to the cell is a primary effect, in the other it is
secondary:
1) Liquid-crystal to gel phase transitions in lipid membranes, possibly due to

gel-phase separation of high melting point phospholipids. This process has
been proposed to lead to the development of chilling injury in sensitive plants
and their organs. Gel phase regions are more fragile, their interfaces with the
liquid crystal zones are leaky, and the remaining liquid crystal zones may be
unstable. All of these will lead to leakiness
2) Low storage temperatures induce dysfunction of enzymes and a

redistribution of cellular calcium. This leads to metabolic changes that
51
Chapter 2
increase the net formation of AOS. These will damage cell membranes by
oxidizing them. Oxidized membranes are more brittle, and the introduction of
hydrophilic peroxy groups into the hydrophobic core of the membrane
disrupts the bilayer structure, which is responsible for the impermeability of
the membrane. The gel phase transition temperature also increases as the
membrane oxidizes – see point one.
In general, an increase in ion leakage is considered to be good evidence for tissue

injury, such as that induced by chilling. It is a simple technique and very widely
employed.
52
Chapter 3
Chlorophyll fluorescence assessment of

chilling injury in ‘Zibdia’ and ‘Hindi Be-
Sennara’ mango varieties
Loay Arafat, Jeremy Harbinson, and Olaf van Kooten
Horticultural Production Chains Group, Marijkeweg 22, 6709 PG, Wageningen

Chapter 3
Abstract
Chlorophyll fluorescence (Fv/Fm ratio) of mango (Mangifera indica L.) fruits was
evaluated as an indicator of chilling injury development during storage. The Fv/Fm
ratio of two mango varieties ‘Zibdia’ and ‘Hindi’ was determined during a 36 day
storage period at temperatures of 2, 4, 7, 10 and 20oC and at different harvest
maturity stages, i.e. immature (M1), half-mature (M2) and fully mature (M3). In
general, 'Zibdia' mango had a higher incidence of chilling injury at low temperatures
than 'Hindi', and the M3 fruits revealed more injury than M1 and M2 fruits in both
mango varieties. However, the initial occurrence of injury after 10 days of storage
was similar for both varieties and it was only after 20 days of storage that it was
possible to identify differences between the cultivars. The Fv/Fm ratio of dark-
adapted fruits decreased rapidly after 12 days of storage in ‘Zibdia’. In 'Hindi' it
decreased gradually until the end of the experiment. However, harvest maturity
stages did not present any differences in Fv/Fm ratio between both varieties. The
correlation of the Fv/Fm ratio with chilling injury was observed for both varieties
during storage. The results of this study suggest that Fv/Fm ratio during storage may
be a good tool to assess chilling injury development before visible symptoms
appear. Zibdia is more sensitive to low storage temperatures than the Hindi cultivar
and the sensitivity varied according to the harvest maturity stages.
Keywords: Chilling injury (CI); Chlorophyll fluorescence (CF); Fv/Fm ratio; Storage; Mango;
Mangifera indica L.
54
Chlorophyll Fluorescence assessment
1. Introduction
Mangoes, like many other tropical and subtropical fruits, are susceptible to
chilling injury (CI) when stored at low temperatures above freezing point. In the
case of mango chilling injury begins at temperatures below 12oC (Chaplin et al.,
1991; González-Aguilar et al., 2000). The most common visual symptoms of CI in
mango fruits are dark skin, a scald-like discoloration, and pitting or sunken lesions
on the peel (Chaplin et al., 1991; Nair et al., 2003); an abnormal ripening of the fruit
and followed by decay is another consequence of storing mangoes at temperatures
below 12oC. The visible symptoms associated with chilling injury depends upon the
plant species, the degree of tissue maturity, and the storage period and
temperature during storage (Wang, 1990). These visible symptoms are the result of
the disruption of normal physiological activity, resulting in metabolic imbalances.
These imbalances, together with the visual symptoms of chilling injury, are the
means with which chilling injury is most frequently quantified and characterized
(Walker et al., 1991).
One of the most sensitive responses of plants to chilling temperatures is the
inhibition of photosynthesis (Allen and Ort, 2001). This inhibition can take several
forms, from damage to photosystems II and I when leaves are illuminated with
normal irradiances at moderately chilling temperatures (Kingston-Smith et al.,
1997), the inhibition of CO2 fixation following moderate chilling in darkness, to the
loss of photosystem II function in darkness under more severe or prolonged chilling
treatments (Allen and Ort, 2001; Kingston-Smith et al., 1997). Chilling-induced
damage to photosystem can be readily determined by using the non-destructive
measurement of chlorophyll fluorescence (CF).
In contemporary research, the most commonly encountered parameter
derived from fluorescence measurements which is used to measure stress
responses is the Fv/Fm ratio. The Fv parameter is equal to the maximum
fluorescence yield (Fm) minus the minimal fluorescence yield (F0), where Fm is the
fluorescence yield when electron acceptors of PSII are reduced to the maximum
level and F0 is the fluorescence yield when electron acceptors of PSII are fully
oxidized. The Fv/Fm ratio of unstressed dark-adapted plants is typically around 0.8
(Björkman and Demmig, 1987; Bolhàr-Nordenkampf et al., 1989) and its value is
proportional to the quantum efficiency of photosystem II electron transport (Maxwell
and Johnson, 2000). Chlorophyll fluorescence has been tested as a tool with which
to assess chilling injury development in green plant tissues; it has many
advantages in this regard as it is rapid, sensitive, non-destructive and it appears
able to detect damage before any visible symptoms appear (DeEll, 1996; DeEll and
Toivonen, 2003). Examples of fruits for which chlorophyll fluorescence has been
55
Chapter 3
used successfully to identify chilling injury are banana (Musa acuminata Colla) and
mango (Mangifera indica L.) (Smillie et al., 1987), cucumber (van Kooten et al.,
1992), green pepper (Capsicum annuum L.) (Lurie et al., 1994) and in lemon fruits
peel (Nedbal et al., 2000). Decreases in the Fv/Fm ratio can be used to measure the
effects of chilling in the dark (Tijskens et al., 1994) and the light (Havaux and
Lannoye, 1984). In older literature other parameters derived from fluorescence
measurements are encountered, for example the decrease of FR (Fi to Fp level of
fluorescence) (Smillie and Hetherington, 1983; Smillie et al., 1987).
The loss of Fv/Fm following chilling in the light and in dark may well be due to
different mechanisms. Under illumination, the loss of Fv/Fm is referred to as
photoinhibition (strictly speaking the photoinhibition of photosystem II), and has
been very extensively studied. It is due to photodynamic damage to the
photosystem II reaction centre, and though the exact mechanism of this damage is
not known, it appears that active oxygen species formed in the reaction centre are
involved (Taylor et al., 1994).
The loss of Fv/Fm that occurs in darkness has been poorly studied and its
mechanism is not understood. However the absence of light excludes the
possibility of a photodynamic process. It is possible that changes in the phase of
the thylakoid lipids may be involved in the dark loss of Fv/Fm even at temperature
above the temperature of the dominant phase transition of the polar lipids of the
thylakoid membrane (Meir et al., 1995; Parkin et al., 1989). A working hypothesis
for the reduction of Fv/Fm in darkness at chilling temperatures is that either thylakoid
membrane oxidation increases (Hariyadi and Parkin, 1991) or that the membrane
transforms to a gel state; either of these processes will result in membrane
dysfunction, and these two processes may act in combination. The loss of Fv/Fm
that ensues is due to a reduction of Fm, not an increase in F0. How this arises is not
well understood in the context of dark chilling, but damage to the oxygen-evolving
complex could explain the phenomenon.
In this article we aim to assess the development of chilling injury in the two
mango varieties that are the most popular in the Egyptian market: Zibdia and Hindi-
Be Sennara. These mangoes were harvested in different maturity stages in order to
assess to what extent maturity stage influences sensitivity to chilling injury. Also,
we intend to verify the possibility of using CF measurements as a tool for detecting
CI sensitivity even before visible CI symptoms are evident. To this end we will use
a range of techniques to measure chilling injury in order to be able to compare
chlorophyll fluorescence with other techniques. Some mango varieties are known to
be more tolerant to low storage temperatures than others (Ibrahim and Khalif,
1999) and Hindi-Be Sennara has been classed as more chilling tolerant than
Zibdia.
56
2. Materials and methods
2.1. Fruit materials
Fruits from two mango (Mangifera indica L.) varieties, Zibdia and Hindi Be-
Sennara, were harvested on 6th August 2002 from trees more than 20 years old
growing in sandy soil. The farm was located in Sharqia, East Egypt (30.35 N and
31.30 E). Zibdia and Hindi Be-Sennara are poly-embryonic varieties. Fruits at three
different harvest maturity stages were harvested from the shaded side of the trees
when the average field temperature was 39oC. The maturity stages were classified
as: immature (M1), half-mature (M2) and fully mature (M3). This classification was
based on the morphological development of fruit shoulder: in M1 fruits the shoulder
is below the stem end, in M2 fruits the shoulder is at the same level as the stem
end, and in M3 fruits the shoulder is above the stem end (Majeed and Jeffery,
2002). The fruits were collected and washed with water at 10 -13oC to reduce both
fruit temperature and the microbial load on the fruit surface. The fruits were then
transported in a refrigerated vehicle to Cairo airport at 10-13oC. The fruits remained
for 21 hours in Cairo airport before being shipped to the Netherlands [a journey of 5
hours]. Following the arrival of the fruits in the Netherlands it took 50 hours to
transfer the fruits to their temperature controlled storage rooms. During this period
the fruits were held either at ambient north European summer conditions or for
short periods at 5○ C (Figure 1). Temperatures were recorded during the transport
process using a miniature data logger (Escort Junior, Escort Data Logging Systems
Ltd, Auckland New Zealand) placed amongst the fruits.
2.2. Storage conditions

Upon arrival in Randwijk, the 360 fruits were divided into 5 batches for non-
destructive measurements (chlorophyll fluorescence and chilling injury index); each
one was composed of 72 Hindi and 72 Zibdia fruits (24 fruits of each maturity stage
in three replicates. Destructive measurements such as ion leakage was measured
on two mango varieties (2160 fruits for Zibdia and 2160 fruits for Hindi ) which were
divided into 5 batches; each one composed 432 fruits (Zibdia 216 and Hindi 216
fruits), with 72 fruits of each maturity stages in three groups as replicates. 9 fruits of
each maturity stage were picked every 5 days intervals for measuring ion leakage.
These batches were stored at 5 different temperatures: 2, 4, 7, 10, and 20oC.
Chlorophyll florescence was measured every 3 days up to 36 days, and the CI
index and ion leakage were measured every 5 days up to 35 days.
57
Chapter 3
Mango varieties were harvested on 10th August 2002
Pre-cooling with cold water 10-13oC
Packaging in carton boxes in one layer
Transferred by refrigerated car 10-13oC
Mango stayed 21 hours in Cairo airport at 5oC for on 10th August
Shipping by plane for 5 hours at 10-13oC on 11th August
Mango stayed 28 hours in Amsterdam airport on 11th August from 8:00 AM up to

12:30 PM 12th August
On 12th August, mango received at fruits station and transferred refrigerated car at
5-7oC
2 hours for distributing mango fruits on treatments according to temperature

cultivars maturity stages
Fruits stayed 22 hours later on 13th August at 5oC
Experiment run on 14th August until 13th September
Figure 1.The logistic chains during three days of import two mango varieties to Netherlands 2002
58
2.3. Chilling injury index

A visual assessment of external damage, such as pitting, water soaked areas,
and decay is often used to assess chilling injury. This approach is obviously
subjective in nature, but frequently employed in routine assessments of product
damage. In order to attempt to relate the more objective measurement techniques
of chlorophyll measurements, electrolyte leakage and chlorophyll fluorescence to
the visual assessment of injury we chose to score visual injury using the method of
Chaplin et al (1991). This method requires that fruit from a batch are scored for
injury according to the following scale: 0 = no damage (ND); 1 = very light damage
(VLD); 2 = light damage (LD) (< 5% area affected); 3 = moderate damage (MD) (6-
25% surface affected); 4 = severe damage (SD) (26-50% surface affected) and 5 =
very severe damage (VSD) (>50% surface affected). The CI index is then
calculated using the following formula:
CI index = (1*FN VLD/FN) + (2*FN LD/FN) + (3*FN MD/FN) + (4*FNSD/FN) +
(5*FNVSD/FN)
Where VLD, LD, MD, SD and VSD were the percentage of fruits presenting
the different degrees of CI and the FN means the total fruits in treatment. The CI
index was measured every 5 days up to a maximum of 35 days.
2.4. Chlorophyll fluorescence

CF data were recorded from individual dark-adapted fruits. Measurements were
collected from four positions per fruit and were made every three days using a
commercial fluorimeter (Mini-PAM, Walz, Effeltrich, Germany) controlled using data
acquisition software supplied with the fluorimeter (Win Control, Walz, Effeltrich
Germany). The parameters recorded were F0 (minimal fluorescence), Fm (the light-
saturated yield of fluorescence) and the Fv/Fm ratio (the ratio of variable
fluorescence to maximum fluorescence, where Fv = Fm -F0). Damage can be
inferred by a decrease in the Fv/Fm ratio below 0.75 - 0.78, which in biophysical
terms implies a reduction in the photochemical conversion efficiency of
photosystem II (Toivonen and DeEll, 2001).
2.5. Chlorophyll content

Samples of 12 fruits were collected at 5-day intervals and the samples were
divided into peel and pulp. Samples were stored at –80oC. To avoid photo-oxidation
of chlorophylls all preparations were carried out in the dark. The peel samples were
frozen at -80oC and lyophilized under a strong vacuum for 7 days. The lyophilized
samples stored in desiccators at room temperature in the dark. The extraction
chlorophyll method of Mónica et al. (1994) was modified by using N,N-
59
Chapter 3
dimethylformamide (DMF) instead of acetone. The samples were then ground to

powder in a ball mill. To 0.8 g of ground powder, 5 ml (DMF) was added. The
suspension was sonicated for 15 min at 4oC and then stored at 4oC for 16 hours to
allow the DMF to leach the pigments from the sample (Minguez-Mosquera et al.,
1991). Finally, 1 ml of the supernatant was centrifuged for 5 min at 16000 rpm and
4oC to remove any suspended material and the clarified supernatant was then
analyzed by HPLC. This system consisted of a P580 pump (Dionex,), a MIDAS
auto-sampler (Spark, Emmen, The Netherlands) and a UVD 340S diode array
detector (Dionex, Sunnyvale, USA), all linked to Chromeleon chromatography
software (Dionex, Sunnyvale, USA). Separations were carried out at 30oC using a
250mm long 4mm diameter LiChrospher 100 RP-18 (5 µm) column (Merck,
Darmstadt, Germany). Chlorophyll was separated with gradient elution at a flow of
1.2 ml min –1, using acetonitrile: methanol: water (84:9:7 v/v) (solvent A) and
methanol: ethyl acetate (68:32 v/v) (solvent B). The gradient was as follows: 0-12
min, 0-100% B; 12-18 min, 100% B (García-Plazaola and Becerril, 1999).
Chlorophyll was detected at 445 nm.
2.5. Ion leakage

Samples of 9 fruits were collected at 5-day intervals. Disks (7 mm diameter) of
peel and pulp tissue were cut from five different parts of each fruit using a cork-
borer. The disks were washed three times in demineralized water and placed in 10
ml 0.4 M mannitol in demineralised water at 24oC for 3h (Hakim et al., 1999).
Electrical conductivity of the aqueous phase was measured using a conductivity
meter, after which the tissue samples were killed by heating in water bath at 100oC
for 20 minutes. This cooking process allows the release of all electrolytes from the
tissue. Once cooled to room temperature the conductivity was re-measured and the
relative electrolyte leakage from the uncooked pulp and peel samples was
calculated as follows:
Conductivi ty after 3 h
IL (%) = x 100
Conductivi ty after boiling
2.6. Statistical analysis

Data for the evolution of CF parameters in term (Fm, F0 and Fv/Fm ratio), CI-
index, chlorophyll and ion leakage in time were analyzed using analysis of variance
(ANOVA), with three factors for temperature, varieties, and harvest maturity stage.
Means were compared using the least significantly differences (L.S.D.) at p=0.05.
Data were analyzed for two periods: the first is from 0-20 days, when all treatments
were present and the second is from day 0-35 days for all treatments except for
60
storage at 20oC, for distractive measurement. Linear regression analysis and

ANOVA were analyzed at the 5% probability level. The statistical software package
Genstat 8 (Lawes Agricultural Trust, Rothamsted Experimental Station, UK) was
used.
3. Results
3.1. Chilling injury index
The chilling injury index, as a function of storage time in days for all the
maturity stages of both varieties of mango at different temperatures is shown in
figure 2. CI index shows a significant interaction at p≤0.05 when the storage time,
temperature, varieties and maturity stages were considered. Considering the
different maturity stages, it is clear that the immature stage (M1) of both varieties is
more resistant to damage compared to the other maturity stages.
At storage temperatures of 2°C and 4°C there was no evidence for CI before
10 days of storage in all the maturity stages and for both the varieties used in this
study. In the case of the fruit stored at 7°C no injury was detected before 20 days.
Storage at 10°C produced no signs of injury within the 35 days of the experiment
irrespective of the cultivar or the maturity stage. The first symptoms that appeared
after 10 days or 20 days were black spots on the peel of the fruit. Though an
increase in storage temperature from 4°C to 7°C increased the time delay before
development of injury, the degree of damage that developed between 10 and 20
days was slight. As a result of this minor development of injury before 20 days
there was only a weak effect of storage temperature on damage development
before 20 days irrespective of the variety or developmental stage.
After 20 days differences emerged between the cultivars and maturity stages
with respect to the temperature dependency of damage. After 20 days of storage CI
developed more rapidly in Zibdia than in Hindi be-Sennara. With increasing
duration of storage it also became clear that chilling at 2°C and 4oC produced the
greatest degree of chilling injury, with the 4°C treatment being more damaging than
the 2°C treatment. At 7oC the degree of injury was in general less severe and no
symptoms injury developed at 10°C and 20oC. The sensitivity of Hindi to injury was
independent of maturity stage, whereas the sensitivity of Zibdia increased as the
fruits became more mature.
3.2. Chlorophyll fluorescence parameters

In figure 3, the Fv/Fm ratio of both mango fruits was plotted as a function of
storage time at different storage temperatures. A significant interaction is found
when temperature and varieties are considered.
61
Chapter 3
6
ZIBDIA HINDI
M1 M1
5
0
6
M2 M2
5
Chilling injury index
0
6
M3 M3
5
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 2. Chilling injury index versus storage time of two mango cultivars Zibdia and Hindi harvested
at different maturity stages: immature (M1), half-mature (M2) and full mature fruits (M3) as a function
of storage time at different storage temperatures. Symbols represent the storage temperatures are
(Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -«-
200C). Chilling injury index expressed as mean (n=3). The Vertical bars indicated to L.S.D. (p=0.05).
62
It is clear that overall there is a significantly greater decrease in the Fv/Fm Zibdia
compared to Hindi. With the exception of the 20°C treatment, the decrease of Fv/Fm
in Hindi appears linear and gradual, with a trend to a greater loss of Fv/Fm at lower
temperatures, though the 4°C treatment always had a lower Fv/Fm than the 2°C
treatment (not statistically significant). The Fv/Fm from the 20°C treatment followed
the same trend as all the other treatments until around day 9 after which it
decreased more rapidly to about 0.45 at days 18 and 21. After day 21 the
measurements on the fruits stored at 20°C ceased owing to their deterioration.
Compared to Hindi, Zibdia showed a different pattern of change of Fv/Fm
against storage time. It is clear that for storage temperatures of 2°C, 4°C and 7°C
Fv/Fm initially decreased in a manner similar to that shown by Hindi, but after a
certain time Fv/Fm began to decrease much more rapidly. This break in the Fv/Fm
temperature relationship occurred at about 21 days for the fruits stored at 2°C and
at about 30 days for the 7°C fruits. In the case of the 4°C fruits the point of the
break is less easy to determine, but it could be as low as 6 days. It is clear that the
4°C storage temperature produces a greater decrease in Fv/Fm than 2°C, though at
no single temperature is this difference significant. The 20°C storage temperature
produced in Zibdia, as it did in Hindi, a rapid loss of Fv/Fm though in this case the
decrease was comparable to that shown by the 4°C treatment. Notably, though at
20°C for a decrease of Fv/Fm to around 0.55 in Zibdia marked the end of the
storage life of the fruit, during chilling a much lower Fv/Fm could be achieved.
Both FM and F0 values increased in both varieties in the initial storage period
up to day 12 (Figure 4), and in the case of both FM and F0 this increase was
significant. Regarding the effect of the storage temperature on F0 values, it was
found that the values increase as the storage temperature decrease regardless of
the type of mango. F0 values change according to the storage period. In the first 10
days and at all the storage temperatures, the values are almost doubled. During the
storage period 12-24 days, F0 decreased gradually almost to the original values,
after which there was no further significant change irrespective of the storage
temperature. Hindi fruits stored at 20oC presented the highest F0 values compared
with fruits stored at temperatures from 2 to 10oC, nevertheless, Zibdia fruits showed
higher increase in F0 in comparison to the fruits stored at 7 and 10oC.
Fm increased significantly (p<0.05) after day 6 and up to day 12 independent
to storage temperatures, followed by a steady decrease until the end of the storage
time (36 days) and the decrease was more marked in Zibdia compared to Hindi.
After the 12 day maximum, larger decrease was observed in Zibdia fruits stored at
63
Chapter 3
2 and 4oc compared to fruits stored at 7 and 10oC. Fruits stored at 20oC showed
decreases in Fm beginning on day 9 and extending to day 21 (Figure 5).
1.0
0.8
0.6
Fv/Fm Ratio
0.4
0.2
ZIBDIA
0.0
1.0
0.8
0.6
0.4
0.2
HINDI
0.0
0 3 6 9 12 15 18 21 24 27 30 33 36
Storage time (days)
Figure 3. Fv/Fm ratio of Zibdia and Hindi of mango cultivars as a function of storage time at different
storage temperatures. Symbols represent the storage temperatures are (Zibdia: -{- 2, -- 4, -U- 7,
-- 10 and -«- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -«-200C). Fv/Fm ratio
expressed as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05.
3.3. Total Chlorophyll AB content and A:B ratio
Figure 5 depicts the variation of total chlorophyll (Chlab) in both varieties as a function of
storage time, at different storage temperatures. The interaction (p<0.001) was
significant among days, temperatures, maturity stages and varieties. Chlab content
decreased with storage duration at the different maturity stages for both varieties. In
comparison to Zibdia, Hindi fruits initially contained less Chlab at all the maturity stages
(~30 vs ~55 mg/100 g-1 DW).
64
ZIBDIA HINDI
1000
800
600
F0
400
200
0
3500
3000
2500
Fm
2000
1500
1000
500
0
0 3 6 9 12 15 18 21 24 27 30 33 36 0 3 6 9 12 15 18 21 24 27 30 33 36
Storage time (days)
Figure 4. Presents the F0 and Fm parameters of two mango varieties Zibdia and Hindi as function of
storage time at different storage temperatures. Symbols represent the storage temperatures are
(Zibdia:-{-, - -4, -U- 7, -- 10 and - ª- 20oC) and (Hindi: -z- 2, -- 4, -c- 7, -- 10 and -ª-
20oC). F0 and Fm expressed as mean (n=3). The verticals bars indicated to L.S.D. at p=0.05
However, the final Chlab content was largely the same in both varieties, with
the exception of the 10°C Zibdia fruits, which retained considerably more
chlorophyll than the other Zibdia fruits. The effect of storage temperature on
theChlab content of Hindi was not significant, except for the 20°C control which lost
chlorophyll more rapidly than the cold stored fruits. In the case of Zibdia there was
a noticeable effect of different low storage temperatures. As noted above, the 10°C
Zibdia retained the most chlorophyll irrespective of the initial maturity stage of the
65
Chapter 3
fruits, the 2°C and 7°C fruits had similar amounts of Chlab, and the least chlorophyll
was present in the fruits stored at 4°C.
For both Zibdia and Hindi fruits stored at 20oC and irrespective of the
maturity stage, Chlab decreased rapidly during storage, faster than in any other
temperature. Other than the higher initial Chlab content, there did not appear to be a
marked effect of maturity stage on the response of Chlab content to storage.
Figure 6 shows the chlorophyll a/b ratio (Chla/b) plotted against the storage time for
both varieties of mango. The initial Chla/b initial values are the same for both
varieties at all the maturity stages. However during storage there are interesting
differences between the two varieties which are independent of maturity stage at
time of harvest. For Zibdia the Chla/b ratio was in nearly all cases (M1 fruits stored
at 4°C and measured at 35 days was the only clear exception) largely unchanged
during the storage. Hindi, on the other hand, showed increases in the Chla/b ratio
after about 15 days of low temperature storage and 10 days at 20oC.
3.4. Ion leakage
Figure 7 shows the ion leakage percentage plotted as a function of storage

time (days) at different storage temperatures for both the peel and the pulp of
Zibdia and Hindi mango fruits at three different maturity ages. It is apparent from
the figure that fruit peel has less ion leakage compared with fruit pulp. In very broad
terms it is clear that ion leakage increases with storage duration, but when
considered in more detail there are some interesting differences between cultivars,
maturity stages, and treatments. As with the other measurements described above,
the responses of the fruits stored at 20°C were generally different to those of fruits
stored at reduced temperatures. With the exception of the Zibdia M3 peel and the
Hindi M1 peel, leakage increased more rapidly from the 20°C fruits than any other
treatment. After 20 days of storage the condition of these fruits became so bad that
no further measurements could be made upon them. For fruits stored at low
temperatures ion leakage had an overall linear or curvilinear increase with time.
Leakage from the peel of either cultivar was not strongly affected by temperature in
maturity stages M1 and M2, but in the M3 stage there was increased temperature
dependency in both cultivars. In the M3 stage it was clear that for both Zibdia and
Hindi 4°C storage produced the greatest increase in leakage at low storage
temperatures, followed by storage at 2°C. In neither cultivar was the initial leakage
from the pulp tissue dependent on maturity stage. For both cultivars and all maturity
stages the least increase leakage of the pulp tissue at the end of the measurement
period was shown by fruits stored at 10°C. The greatest leakage was in most cases
provoked by storage at 4°C, or more rarely 2°C. Where the greatest damage was
caused by 4°C storage, the next most severe damage was caused by storage at
66
2°C. Maturity stage at harvest had no clear effect on the progression of the
increase in leakage during storage.
3. Discussion
The chlorophyll fluorescence based Fv/Fm parameter has, along with other
parameters derived from fluorescence or other biophysical probes of
photosynthesis, a long history as a tool for assessing stress and damage in green
plant tissues. The results obtained in this study show that in Zibdia, and to a lesser
extent in Hindi, cold storage produces changes in Fv/Fm that are temperature
dependent (Figure 3). Thus according to changes in Fv/Fm parameter Zibdia is
more chilling sensitivity than Hindi. Thus, this simple fluorescence parameter is
able to detect temperature induced damage that develops in darkness in
photosystem II. The changes in Fv/Fm cannot be substantially attributed to an
increase in F0; though F0 increases with time of storage, so does Fm, and the
increase in F0 is insufficient to account for the large losses of Fv/Fm.
Changes in the absolute values of fluorescence yield are difficult to interpret
whenever these changes occur simultaneously with changes in chlorophyll content.
However, figures 3 and 4 do give some insights into the underlying causes of
changes in Fv/Fm in terms of F0 and Fm (Figure 8 and 9). Under low temperature
conditions, the decreases in Fv/Fm of Hindi fruits are correlated with small increases
in F0 and larger increases in Fm. Such a small increase in F0 is frequently observed
during the decreases of Fv/Fm that occur in the early stages of light-induced
photoinhibition experiments on photosystem II. At 20°C, Hindi fruits behave in a
similar way as they do under cold-storage, except that the changes in F0 and Fm
are more extreme (Figure 4). Zibdia fruits at 20°C behave in a similar way to Hindi
fruits, but under cold storage their behaviour is more complex. The decreases in
Fv/Fm that occur in Zibdia fruits are larger than encountered with Hindi fruits, and
these large decreases are closely correlated with decreases in Fm (Figure 4), which
is consistent with the results obtained from Hindi. The relationship between F0 and
Fv/Fm from Zibdia is more complex than that found with Hindi; at 10°C and 7°C the
responses are similar to Hindi, but at 4°C and 2°C some samples show a response
similar to Hindi whereas others show large decreases of Fv/Fm that are independent
of changes in F0, so overall there is no correlation between F0 and Fv/Fm. Based on
the visual assessment of chilling injury (the CI index) the following conclusions can
be drawn:
• Initially (M1 stage) Hindi and Zibdia have an equal degree of chilling
tolerance
• With increasing maturity Zibdia becomes more chilling sensitive than Hindi
67
Chapter 3
• In all cases the greatest degree of chilling injury developed at 4°C and not
2°C.
• Storage at 10oC produced no signs of injury
Increases in the degree of sensitivity to the development of visual symptoms of
injury can occur as mango fruits mature (Majeed and Jeffery, 2002), but the extent
to which this happens varies from cultivar to cultivar. This would appear to be an
important physiological property from the point of view of both breeding and
selecting the best cultivars when storage is required. Also, at least as far as
deterioration of the visual appearance of the fruit is concerned, relatively prolonged
storage at 10°C is possible, but below this temperature damage will develop with
decreasing temperature (González-Aguilar et al., 2000; Nair et al., 2003). Based
on the changes to the visible appearance of the fruits, the report that Zibdia is more
chilling sensitive than Hindi (Ibrahim and Khalif, 1999), is only correct at later
maturity stages; or put another way, if harvested early Zibdia is as chilling tolerant
as Hindi. It is surprising that the lowest temperature did not produce the greatest
degree of injury; this pattern was also observed in the responses of Fv/Fm,
chlorophyll content (in Zibdia) and, to a lesser degree, electrolyte leakage so it is a
general phenomenon. A simple interpretation of this response is that though low
temperatures produce chilling injury, the development of that damage is
temperature dependent (Majeed and Jeffery, 2002). In the case of mango fruit this
could result in less damage developing at 2°C compared to 4°C.
According to the changes in FV/FM Zibdia is more chilling sensitivity than Hindi. The
CI-index data suggest that Zibdia only in the M2 and M3 stagesis more chilling
sensitivity than Hindi. Other measurements of chilling injury do not, on the whole,
strongly support the proposition that Hindi is more chilling tolerant than Zibdia.
Though Hindi looses its chlorophyll more slowly than Zibdia, Zibdia has much more
chlorophyll to start with. Because of the relatively good retention of chlorophyll in
the 10°C Zibdia fruits of all maturity stages, this cultivar has a relatively greater
variation in its chlorophyll retention than does Hindi. It is not clear, however, that
this means that Zibdia is more tolerant than Hindi. The degree of ion leakage from
either the pulp or the peel also does not indicate any greater chilling tolerance in
Hindi compared to Zibdia. These different results raise problem for the
determination of chilling tolerance. Depending on the technique chosen, the results
differ in detail. Some techniques, for example CI-index, reveal interesting variation
in chilling tolerance that could be of value in breeding
68
100
ZIBIDA HINDI
M1 M1
80
60
40
20
0
100
Total chlorophyll A and B (mg 100 g-1 DW)
M2 M2
80
60
40
20
0
100
M3 M3
80
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)

Figure 5. Total chlorophyll A and B (Chlab) content of Zibdia and Hindi mango cultivars which were
harvested at different maturity stages: immature (M1), half-mature (M2), and fully mature fruits (M3)
versus storage time at 5 different storage temperatures. Symbols represent the storage
temperatures are (Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: - z - 2, - - 4, -
▲- 7, -¡ 10 and -«-200C). Chlb content expressed as mean (n=3). The vertical bars indicated to
L.S.D. at p=0.05 for two period (0- up to 20 days) when all treatments were present and the second
is from (0- up to 35 days) for all treatments except for storage at 20oC.
69
Chapter 3
3.0 ZIBDIA HINID

M1 M1
2.5
2.0
1.5
1.0
0.5
0.0
3.0
M2 M2
Chlorophyll A: B ratio (mg 100 g-1 DW)
2.5
2.0
1.5
1.0
0.5
0.0
3.0
M3 M3
2.5
2.0
1.5
1.0
0.5
0.0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)

Figure 6. Chlorophyll A:B ratio of Zibdia and Hindi mango cultivars harvested at different maturity
stages: immature (M1), half-mature (M2) and fully mature fruits (M3) versus storage time at 5
different storage temperatures. Symbols represent the storage temperatures are (Zibdia: -{- 2, --
4, -U- 7, -- 10 and -«- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -«-200C). The ratio
expressed as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05 for two period (0- up to 20
days) when all treatments were present and the second is from (0- up to 35 days) for all treatments
except for storage at 20oC.
70
PEEL
100 ZIBDIA HINDI
M1 M1
80
60
40
20
100
Ion leakage % of fruit peel
M2 M2
80
60
40
20
100
M3 M3
80
60
40
20
0
0 5 10 15 20 25 30 350 5 10 15 20 25 30 35
Storage time (days)
Figure 7a. Ion leakage percentage of fruits peel versus storage time of two mango varieties Zibdia
and Hindi harvested at different maturity stages: immature (M1), half-mature (M2), and full mature
fruits (M3) as a function of storage time at different storage temperatures. Symbols represent the
storage temperatures are (Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: - z - 2, -
- 4, -▲- 7, -¡- 10 and -«-200C). CO2 expressed as mean (n=3). The Vertical bars indicated to
71
Chapter 3
PULP
120 ZIBDIA HINDI
M1 M1
100
80
60
40
20
0
120
M2 M2
Ion leakage % of fruit pulp
100
80
60
40
20
0
120
M3 M3
100
80
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)

Figure 7b. Ion leakage percentage of fruits pulp versus storage time of two mango varieties Zibdia
- 4, -▲- 7, -¡- 10 and -«-200C). CO2 expressed as mean (n=3). The Vertical bars indicated to
72
programs: in Hindi chilling tolerance is independent of maturity stages, whereas for

Zibdia it decreases with increasing maturity. This pattern is not clearly revealed by
other assays. This implies that case needs be taken when comprising chilling
tolerances that have been evaluated by different techniques. It may be, however,
that a more detailed analysis of the results obtained with the different assays will
reveal patterns that are not evident in the time dependencies of the assays.
In qualitative terms the measures of chilling injury support each other, a quantitative
comparison reveals a more complex relationship. A comparison of the CI-index with
Fv/Fm (Figure 10) shows that there are three classes of relationship.
Fruits stored at 10°C show a vertical distribution of points, which reflects the
small decrease of Fv/Fm at 10°C coupled with the lack of change in CI-index. Under
these conditions the Fv/Fm measurement reveals damage to PSII that is not
paralleled by changes in the CI-index. The second type of response is found at all
other temperatures for Hindi and at 7°C for Zibdia. Here there is a large increase of
the CI-index data which is correlated with only a small change in Fv/Fm, so Fv/Fm is
nearly independent of CI-index. The third class of response is found only at 2°C
and 4°C for Zibdia. Here there is a good correlation between CI-index and Fv/Fm.
These correlations, or lack of them, imply that if CI-index is taken to be a good
yardstick for measuring CI then Fv/Fm is a poor tool for measuring injury except in
certain limited cases.
This pattern of results is also found in the relationship between Fv/Fm and both total
chlorophyll ab content and electrolyte leakage (Figures 11 and 12). In this Fv/Fm
only correlates well with ion leakage and chlorophyll content in Zibdia at 2°C and
4°C. The correlations obtained between Fv/Fm and electrolyte leakage for the
control, un-chilled fruits of Zibdia and Hindi are, however, good, and for chlorophyll
a content and Fv/Fm, reasonably good. So, on the one hand it is clear that changes
in Fv/Fm are not, in general, a good indicator of chilling injury in dark-stored mango
fruits except under relatively severe treatments of a susceptible cultivar. On the
other hand, however, it seems that changes in Fv/Fm could be used just as well as
changes in either chlorophyll content or electrolyte leakage to monitor the normal
ripening process that occurred at 20°C (Jacobi et al., 1998). In conclusion, the
generally poor correlation that exists between Fv/Fm and other measures of change
during chilling conflicts strongly with other results that support the use of Fv/Fm as a
measure of chilling injury (Meir et al., 1995). In contrast to the generally poor
correlation that was found between Fv/Fm and CI-index, the relationship that exists
between CI-index and electrolyte leakage from either the pulp or peel is good
(Figures 10 and 12). Likewise, the relationship between CI-index and total
chlorophyll content (Figure 13) is very close, though it differs between varieties
73
Chapter 3
0.90 ZIBDIA HINDI

o o
2 C 2 C
0.60
0.30
y = -0.0002x + 0.7932
y = 8E-05x + 0.5764
R 2 = 0.0867
R2 = 0.0032
0.00
0.90
o
4 C 4 C
o
0.60
0.30
y = -0.0002x + 0.7981
y = 0.0002x + 0.4201
R 2 = 0.1273
R 2 = 0.0232
0.00
0.90
o o
7 C 7 C
0.60
Fv/Fm ratio
0.30
y = -3E-05x +0.7286 y = -6E-05x + 0.7774
R2 = 0.0027 R 2 = 0.0199
0.00
0.90
o o
10 C 10 C
0.60
0.30
y = -8E-05x + 0.808 y = -0.0001x + 0.8271
R 2 = 0.1281 R 2 = 0.1133
0.00
0.90
o o
20 C 20 C
0.60
0.30
y = -0.0005x + 0.9348
y = -0.0007x + 0.9877
R2 = 0.3706 R 2 = 0.4262
0.00
0 300 600 900 0 300 600 900
F0 parameter
Figure 8. The correlation Fv/Fm ratio parameter in function of F0 of tow mango cultivars ‘Zibdia’ and
‘Hindi’ which stored at different storage temperatures (2, 4, 7, 10 and 20oC) for 36 days. The lines
represent linear regressions on the data.
74
ZIBDIA HINDI
0.90
o
2 C 2 C
o
0.60
0.30
y = 0.0002x + 0.2103 y = 8E-05x + 0.5704
R 2 = 0.6187 R 2 = 0.1743
0.00
0.90
o o
4 C 4 C
0.60
0.30
y = 0.0003x + 0.1195 y = 1E-04x + 0.5211
R 2 = 0.713 R 2 = 0.2862
0.00
0.90
o o
7 C 7 C
0.60
0.30
y = 7E-05x + 0.5924
R 2 = 0.2334 y = 4E-05x + 0.6732
Fv/Fm ratio
R 2 = 0.1723
0.00
0.90
o
10 C 10 C
o
0.60
0.30
y = 7E-06x + 0.7519 y = 3E-05x + 0.7098
R 2 = 0.0131 R 2 = 0.0902
0.00
0.90
o
20 C 20oC
0.60
0.30
y = 0.0002x + 0.4164 y = 0.0003x + 0.1898
R 2 = 0.3503 R2 = 0.5378
0.00
0 1000 2000 3000 0 1000 2000 3000
Fm Parameter
Figure 9. Present the correlation Fv/Fm ratio parameter in function of Fm of tow mango cultivars
‘Zibdia’ and ‘Hindi’ which stored at different storage temperatures (2, 4, 7, 10 and 20oC) for 36 days.
The lines represent linear regressions on the data.
75
Chapter 3
owing to the greater size of the changes in chlorophyll content that occur in Zibdia
compared to Hindi. This problem of processes of change occurring over different
ranges of values has been addressed by the batch-analysis approach of Schouten
et al (2002). It seems reasonable to propose that this method could be adapted to
eliminate the problem of differing initial chlorophyll contents complicating the use of
a chlorophyll content measurement to assess chilling damage development during
mango storage. Comparing different developmental stages from both Zibdia and
Hindi subjected to different chilling treatments, there is always a similar, consistent
positive correlation between CI-index and ion leakage from either the pulp or peel.
It seems therefore that CI-index, electrolyte leakage, and changes in chlorophyll
content are a homogeneous group of measurements for chilling injury. Overall the
correlations are unaffected by maturity stage or temperature. The match is not,
however, perfect; for example in Zibdia chilled at 4°C there is an influence of
maturity stage on the relationship between leakage from the peel and CI-index
(Figure 14a), just as there is at 4°C and 7°C for leakage from the pulp of Zibdia and
CI-index (Figure 13b).
The Fv/Fm parameter responds quantitatively differently to the members of
this group, though in qualitative terms it does still offer a means of identifying
chilling damage. Of course all of these measurements are physiological in nature
and do not say anything about product flavour etc. However in spite of this last
reservation, it might be worth exploring tools for chilling injury assessment based
upon colorimetric analysis, image analysis, or bio-impedance measurements as
this measures are based upon process that are measured by the CI-index,
chlorophyll content and ion leakage technique, or to explore more sophisticated
applications of fluorescence.
The Chla/b ratio responds differently to chilling in the two cultivars
investigated. In the case of Zibdia the relatively constant ratio that is maintained
during large reductions of chlorophyll content implies that the loss of chlorophyll is
evenly distributed across all components, whereas with Hindi the increasing ratio
suggests a preferential breakdown of Chlb containing chlorophyll binding proteins,
such as the LHC of photosystem I or II (DeEll and Toivonen, 2000; Rosenqvist and
van Kooten, 2003).
Acknowledgement
The author would like to appreciate the Egyptian High Educational Ministry
for support the PH.D., full program. Also Dr. Ep Heuvelink for statistical analysis
advice.
76
ZIBDIA HINDI
1.0
o o
2 C 2 C
0.8
0.6
0.4
0.2
y = -0.1288x + 0.8697 y = -0.0411x + 0.7857
R2 = 0.6301 R2 = 0.3912
0.0
1.0
o o
4 C 4 C
0.8
0.6
0.4
0.2
y = -0.1296x + 0.8225 y = -0.0333x + 0.7629
R2 = 0.7576 R2 = 0.4761
0.0
Fv/Fm Ratio
1.0
7oC o
7 C
0.8
0.6
0.4
0.2 y = -0.0337x + 0.7765 y = -0.0133x + 0.7792

R2 = 0.443 R2 = 0.1028
0.0
1.0
o
10 C 10 C
o
0.8
0.6
0.4
0.2
0.0
0 1 2 3 4 50 1 2 3 4 5
Figure 10.The correlation Fv/Fm ration in function of CI-index of Zibdia and Hindi at different storage
temperatures for 35 days (Zibdia: -{- 2, -- 4, -U- 7, -- 100C) and (Hindi: - z - 2, - - 4, -▲- 7,
-¡- 100C). Solid lines represent linear regressions.
77
Chapter 3
1.0
ZIBDIA HINDI
o o
2 C 2 C
0.8
0.6
0.4
0.2 y = 0.0081x + 0.3275 y = 0.0073x + 0.574

R2 = 0.5117 R2 = 0.4102
0.0
1.0
o o
0.8 4 C 4 C
0.6
0.4
y = 0.0098x + 0.5086
0.2 y = 0.0102x + 0.2045
R2 = 0.7362
R2 = 0.7925
0.0
1.0
o o
7 C 7 C
0.8
0.6
0.4
Fv/Fm Ratio
0.2 y = 0.0021x + 0.6427 y = 0.0026x + 0.7042

R2 = 0.3589 R2 = 0.1305
0.0
1.0
o o
10 C 10 C
0.8
0.6
0.4
0.2 y = 0.0011x + 0.7154 y = 0.0027x + 0.716

R2 = 0.1484 R2 = 0.3468
0.0
1.0
o o
20 C 20 C
0.8
0.6
0.4
0.2 y = 0.0057x + 0.4933 y = 0.0162x + 0.382

R2 = 0.6519 R2 = 0.5212
0.0
0 20 40 60 80 0 10 20 30 40
Chlorophyll AB content (mg 100 g-1 DW)
Figure 11. The correlation Fv/Fm ratio in function of total chlorophyll AB content of two mango cultivars Zibdia
(A) and Hindi (B) stored at different storage temperatures for 35 days, (Zibdia: -{- 2, -- 4, -U- 7, -- 100C
and -ª-) and (Hindi: - z - 2, - z - 4, -▲- 7, -¡- 100C and -ª-). Solid lines represent linear regressions on the
data.
78
0.9 ZIBDIA HINDI

o o
2 C 2 C
0.6
0.3
y = -0.0085x + 0.9504 y = -0.0027x + 0.8146
R 2 = 0.7643 R 2 = 0.346
0.0
0.9
o o
4 C 4 C
0.6
0.3
y = -0.005x + 0.7875 y = -0.0018x + 0.7685
R 2 = 0.3945 R 2 = 0.5224
0.0
0.9
o o
7 C 7 C
0.6
0.3
Fv/Fm Ratio
y = -0.0011x + 0.7713
y = -0.0004x + 0.7757
R 2 = 0.3807 R2 = 0.0809
0.0
0.9 o o
10 C 10 C
0.6
0.3
y = -0.0004x + 0.7845 y = -0.0008x + 0.8047
R 2 = 0.1042 R 2 = 0.3175
0.0
0.9
o o
20 C 20 C
0.6
0.3
y = -0.005x + 0.9152 y = -0.0069x + 0.9497
R 2 = 0.8149 R 2 = 0.8217
0.0
0 20 40 60 80 100 0 20 40 60 80 100
Ion Leakage % of fruit peel
Figure 12. The correlation Fv/Fm in function of ion leakage percentage of two mango cultivars stored
at different storage temperatures for 35 days. (Zibdia: -{- 2, -- 4, -U- 7, -- 100C) and (Hindi: -
z - 2, - z - 4, -▲- 7, -¡- 100C). Solid lines represent linear regressions on the data.
79
Chapter 3
ZIBDIA HINDI
120
o o
2 C y = -19.58x + 91.966 2 C
R2 = 0.8919 y = -8.7682x + 42.043
R2 = 0.7257
80 y = -9.5242x + 52.032
R2 = 0.787
y = -9.3292x + 48.239
40 R2 = 0.8013
y = -18.88x + 100.94
R2 = 0.815
y = -21.248x + 102.38
R2 = 0.9294
0
Total chlorophyll content (mg 100 g-1DW)
120
4 C
o y = -19.694x + 90.161 4 oC y = -6.3536x + 39.66
R2 = 0.8913 R2 = 0.7141
y = -6.881x + 49.602
80 y = -18.723x + 100.79 R2 = 0.7743
R2 = 0.8925
y = -6.667x + 45.309
R2 = 0.7721
40
y = -19.527x + 96.376
R2 = 0.8769
0
120
7o C y = -21.895x + 92.369
o
7 C y = -8.2527x + 44.498
2
R = 0.8447 R2 = 0.7283
y = -8.1414x + 47.754
80
R2 = 0.6665
y = -22.011x + 97.606
2
R = 0.8547
y = -7.3125x + 50.656
40 R2 = 0.5765
y = -20.164x + 100.61
2
R = 0.8177
0
0 1 2 3 4 5 0 1 2 3 4 5
Figure 13 The correlation of total chlorophyll (A and B) content in function of chilling injury index of
two mango varieties fruits of Zibdia and Hindi varieties were harvested in different maturity stages
(Zibdia: -{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3), which stored at
different storage temperatures (2, 4, 7, 10 and 20oC), over 35 days. Solid lines represent linear
regressions on the data.
80
PEEL
ZIBDIA HINDI
100
o
2oC 2 C
80 y = 14.962x + 12.06
y = 10.636x + 14.145
R2 = 0.9044
R2 = 0.9062
60
40 y = 15.089x + 11.567
y = 20.381x + 3.2427 R2 = 0.8976
R2 = 0.9245
20 y = 10.081x + 18.164
y = 14.979x + 10.1 R2 = 0.8351
R2 = 0.9356
0
100
o
4 C o
4 C
y = 8.9154x + 21.367
80 R2 = 0.7474
y = 19.553x + 3.1611
R2 = 0.8807
60
y = 17.151x + 1.7672
40 R2 = 0.8967
y = 13.448x + 11.22
R2 = 0.8806
20
y = 19.048x + 18.981 y = 16.787x + 6.4412
R2 = 0.6904 R2 = 0.8729
0
100
o
7 C 7oC
80 y = 25.995x - 1.4528
R2 = 0.8508
y = 27.009x + 2.7084
60
R2 = 0.8851
y = 17.938x + 5.888
40
y = 26.558x + 1.0711 R2 = 0.8996
R2 = 0.8161
20
y = 24.624x + 6.3754 y = 27.974x + 1.264
R2 = 0.7735 R2 = 0.9261
0
0 1 2 3 4 5 0 1 2 3 4 5
Figure 14a. Presents the linear relationship in ion leakage percentage of fruit peels in function of
chilling injury index of fruits of Zibdia and Hindi varieties were harvested in different maturity stages
regressions on the data
81
Chapter 3
PULP
ZIBDIA HINDI
100
y = 13.448x + 22.591
o
2 C 2oC R2 = 0.8694
80
60
y = 22.18x + 10.716
R2 = 0.8819
40 y = 15.293x + 27.468
y = 21.468x + 14.544
R2 = 0.7467
R2 = 0.9817
20
y = 16.954x + 17.059 y = 10.811x + 27.716
R2 = 0.9472 R2 = 0.8353
0
100
4 oC o
4 C
80
60
y = 12.089x + 32.325
y = 11.701x + 25.076
2
R2 = 0.7797
40 R = 0.8291
y = 9.4082x + 26.434
y = 14.538x + 21.133 R2 = 0.8369
2
20 y = 16.922x + 26.553
R = 0.7292
2
y = 14.745x + 19.467
R = 0.8644
R2 = 0.9346
0
100
o
7 C 7oC
80 y = 18.751x + 21.219
R2 = 0.8711
y = 19.918x + 22.556
60
R2 = 0.8212
40 y = 9.5394x + 30.9 y = 19.53x + 22.328

R2 = 0.7396
R2 = 0.8173
20 y = 21.48x + 18.015
y = 20.442x + 24.79
R2 = 0.8351
R2 = 0.7206
0
0 1 2 3 4 5 0 1 2 3 4 5
Figure14b. Presents the linear relationship in ion leakage percentage of fruit pulp in function of
chilling injury index of fruits of Zibdia and Hindi varieties were harvested in different maturity stages
regressions on the data
82
Chapter 4
Changes in the concentration of water and

lipid-soluble antioxidants during cold storage
of Zibdia’ and ‘Hindi Be-Sennara’ mango fruits
Horticultural production Chains Group, Marijkeweg 22, 6709 PG Wageningen

University. The Netherlands
Chapter 4
Abstract
The predominant water (ascorbic acid (ASC)) and lipid soluble (α-Tocopherol (α-
TOC) and β-Carotene (β-CAR)) antioxidants (WSA and LSA, respectively) of two
mango varieties (Mangifera indica L. ‘Zibdia ‘and ‘Hindi Be-Sennara) were
measured in the peel and the pulp of fruits stored at temperatures of 2 - 20oC for 35
days. The initial amounts of WSA at the time of harvest were found to differ
according to the maturity stage of the fruit. The WSA content also decreased during
storage dependent on maturity stage at harvest. Zibdia had a higher initial ASC
content than Hindi and had a higher content in for M1 and M2 fruit relative to M3
fruits in both peel and pulp. For the LSA, the patterns of change were more
complex. In the peel of Hindi the initial level of α-TOC increased with increasing
maturity stage, but decreased during cold storage, and the same pattern was found
in Zibdia except that the initial level of α-TOC was lower than for Hindi. In the pulp
of Hindi the α-TOC increased strongly with maturity stage and decreased during
storage, whereas with Zibdia the α-TOC hardly changed with maturity stage. β-CAR
contents that increased by increasing fruit maturity in both Zibdia and Hindi and
also increased during storage, even at 2°C. These results were pronounced even
before visible CI symptoms. Storage Zibdia fruits at 10oC can be considered as the
optimum storage temperature for however, 7oC is optimum for Hindi in terms of
preserving anti-oxidant levels
Keyword: Ascorbic acid (ASC); α-Tocopherol (α-TOC); β-Carotene (β-CAR); Mango fruits
84
Water and lipid-soluble antioxidants
1. Introduction
Fruits and vegetables contain many different water (WSA) and lipid-soluble
antioxidants (LSA), e.g. ascorbic acid (ASC), α-Tocopherol (α-TOC) and β-
Carotene (β-CAR). Epidemiological studies suggest that a high consumption by
humans of fruits and vegetables is associated with numerous health benefits such
as a lower incidence of various degenerative diseases, reduced cardiovascular
disease, reduced risk of cancer, and enhanced immune system, reduced levels of
cell injury and an improved ability to detoxify contaminants and pollutants, (Ching
and Mohamed, 2001; Feskanich et al., 2000; Haegele et al., 2000; Leong and Shui,
2002; Michets et al., 2000; Wang and Jiao, 2000). WSA and LSA are considered to
protect tissues against active oxygen species (AOS), including superoxide radicals
(O2●-), hydrogen peroxide (H2O2), hydroxyl radicals (OH●) and singlet oxygen (1O2)
(Heinonen and Meyer, 1998; SatueGracia et al., 1997). It is thought the
epidemiologically demonstrated health benefits of fruit and vegetables are due at
least in part to the LSA and WSA that they contain.
Just as LSA and WSA are thought to protect against AOS in animals, then
they act in the same way in plant cells where AOS are formed in different
organelles (Hodges et al., 2004). Several studies have outlined the effect of
excessive levels of AOS formation in the plant cell. They produce increases in
oxidative reactions that result in biochemical and physiological degradation of the
lipids and proteins of the cell membrane (Purvis, 2003). Practically, many storage
disorders of fruits are associated with oxidative damage (Purvis, 2004).
The health benefits of humans of the naturally occurring LSA and WSA
found in vegetables and fruits have led to these substances being significant
determinants of the compositional quality of these products. The LSA and WSA
content of a product can differ for many reasons, such as maturity stage at
harvest, the variety, geographic or climatic effects, and post-harvest processing
and storage conditions (Adriana and Delia, 1998). In the case of mango varieties
the maturity stage of fruit at harvest is important: ASC was reported to decrease
with increasing fruit maturity whereas α-TOC and β-CAR increase with increasing
fruits maturity (Lee and Kader, 2000). Recently, it was reported that significant
changes in α-TOC content which increased during ripening of fruits apple. For
example, delayed harvesting of apple raised α-TOC content in the peel, though this
decreased during postharvest processing, e.g. storage and packaging (Barden and
Bramlage, 1994), The β-CAR content has also been reported to increase during the
ripening and ageing of mango fruits (Exotic Fruits, 2000).
Munne-Bosch and Alegre (2002) have summarized the role that ASC plays
in plant cells. It participates in a wide range of metabolic processes including
85
Chapter 4
photosynthesis, photoprotection, the cell cycle, cell-wall growth expansion,

synthesis of ethylene, gibberellins, anthocyanins, and hydroxyproline, and
resistance to environmental stress. An important role for ASC is the detoxification
of superoxide, hydrogen peroxide and singlet oxygen by means of a series of
redox reactions involving which also involve glutathione (Foyer et al., 1991). α-TOC
is the major lipid-soluble antioxidant in bilayer membranes, which it protects against
singlet oxygen, peroxyl radicals and nitrogen oxide species (Christen et al., 1997).
In this way it stabilizes the cell membrane (Shewfelt and Del Rosario, 2000) and
thus prevents damage of tissues arising from membrane dysfunction (Combs,
1992). The carotenoids, in general have two main anti-oxidant roles: they can act
as radical scavengers and in photosynthetic tissues they are also a quencher of
triplet-excited states of chlorophyll a (Hideg et al., 1998); (Mortensen and Skibsted,
1997) Carotenoids scavenge free-radicals in two ways First they can react with
radicals to form stable molecules, and second they can reduce the radical, forming
a stable carotenoidic radical cation from which a carotenoid can be regenerated.
The aim to this study is to measure how WSA and LSA change during storage at
different temperatures in term of developmental changes of two mango varieties
are harvested on different maturity stages. In addition to providing information
about how storage will affect the nutritional value of the mangoes, we also intend to
correlate the changes in anti-oxidant levels with the development of oxidative injury
in the mango fruit and thus examine the possibility that changes in fruit anti-oxidant
levels are involved in the development of the symptoms of chilling injury via
increase in oxidative damage to cell lipids and protein.

2.1. Fruits
Fruits from two mango varieties ‘Zibdia’ and ‘Hindi Be-Sennara’, were harvested,
and imported from Sharqia province, East Egypt (30.35 N and 31.30 E). The
harvesting and logistic chains were described before in chapter 2. day zero on the
graphs was day on which the first measurement was made:
2.2. Storage conditions

The fruits were divided into 5 batches; each one of which was composed of
300 Hindi and 300 Zibdia (100 fruits of each maturity stage). These batches were
stored in 5 different containers at 5 different temperatures: 2, 4, 7, 10 and 20oC.
Twelve fruits were collected from each container every 5 days (up to 35 days) from
which samples of peel and pulp were removed and stored at –80oC until required
for analysis of ASC, α-TOC and β-CAR.
86
2.3. ASC content

To exactly an weighed 5 g sample, 5 ml of 9.5 % w/v oxalic acid, 5 ml
methanol, and 35 ml Milli-Q water were added (Veltman et al., 2000). The mixture
was homogenized for 1-2 min before being filtered 4oC in darkness to avoid photo-
oxidation of ASC. The filtrate was injected directly into the HPLC system. The
sample temperature was kept at 4oC in the automated sampler of the HPLC. The
HPLC system comprised of a Dionex model P580 pump (Dionex, Sunnyvale, USA),
a Lichrospher® 100 RP-18 (5 cm) Lichro CART® 250-4 column (Merck, Darmstadt,
Germany) and a Dionex UVD 3405 detector (251 nm). The mobile phase used was
2.5 g tetrabutylammoniumhydrogensulfate and 55 ml methanol dissolved in 942.5 g
Milli-Q water. The flow rate was kept at 1 ml min-1 and the analyses were
completed within 15 min.
2.5. Protein carbonyl assay

Protein carbonyl grope was measured according to (Levine et al., 1994).
Precisely weighed mango samples (peel or pulp) of about 2.5 g were ground in a
mortar and mixed with 10 ml of 20 mM potassium phosphate buffer (pH 7.0) to
extract soluble proteins. The homogenates were then centrifuged at 16000 rpm for
5 min. One ml samples of the supernatant, to which 500 µl of 10 mM
2,4-dinitrophylhydrazine in 2 M HCl had been added, were allowed to stand at room
temperature for 1 hour, with vortexing every 10-15 min. 500 µl of 20%
trichloroacetic acid was then added and the mixture centrifuged at 11000g for 3
min. The supernatant was removed and the precipitate washed 3 times with 1:1 v/v
ethanol/ethyl acetate mixture to remove any un-reacted dinitrophenol. The sample
was then allowed to stand for 10 min before centrifugation after which the
supernatant was discarded. The washed precipitated protein was then allowed to
dissolve in 0.6 ml guanidine solution (6 M) for 15 minutes at 37°C, after the mixture
was centrifuged to remove any undissolved material. Afterwards, the spectrum was
measured spectrophotochemically against the complementary blank in case of
cured (without sample) samples or against water in case of purified proteins. The
carbonyl content of the protein was calculated from the absorbance of the
dinitrophenylhydrazone measured at 390nm and assuming an extinction coefficient
of 22000 M-1 cm-1.
2.4. α-TOC and β-CAR extraction
To avoid photooxidation of CAR the α-TOC and β-CAR were extracted in

darkness and, all extracts were stored in brown bottles. The samples of peel and
pulp were dehydrated at -80oC in a freeze-drier for 7 days after which they were
stored in a desiccator at room temperature. To extract the α-TOC and β-CAR the
87
Chapter 4
freeze-dried samples were first pulverized in a ball–mill. To 0.8 g of this powder 5

ml N,N-dimethylformamide (DMF) was added. The powder/DMF suspension was
sonicated for 15 min at 4oC (Minguez-Mosquera et al., 1991) and then stored at
4oC for 16 hours to allow complete extraction of the α-TOC and β-CAR (Mónica et
al., 1994). Finally, 1 ml of the suspension was centrifuged for 5 min at 16000 rpm at
4oC to remove all solids and the supernatant solution was then injected into the
HPLC system.

Exactly weighed 2.5 g mango (peel or pulp) samples were ground in a
mortar and mixed with 25 ml of 5% (w/v) metaphosphoric acid, 500 µl of 2% (w/v)
butylated hydroxyltoluene in ethanol, and finally homogenized by a mixer. The
homogenates were filtered and centrifuged at 15000 rpm for 20 min. Then
chromogen was formed by mixing 1 ml of the supernatant solution, 100 µl of 2%
(w/v) butylhydroxytoluene, 0.50 ml of 1% (w/v) TBA (thiobarbituric acid) in 50 mM
NaOH and 0.50 ml of 25% (v/v) HCI, and incubating the reaction mixture at 95oC
for 30 min. The reaction blank was prepared by replacing the sample with
extraction medium and the control for each sample were prepared by replacing
TBA with 50 mM NaOH. The calibration curves made by measuring 1,1,3,3-
tetraethyoxypropane (Sigma) in the range 0-2 mM (TBARS) which was equivalent
to 0-1 mM malondialdehyde (MDA). Tetraethyoxypropane is stoichiometrically
converted into MDA during the acid-heating step of the assay (Iturbe-Ormaetxe et
al., 1998). The amount of TBARS present is expressed as MDA equivalents.
2.7. Assay of protein content

The total protein content in the peel and the pulp of the mango fruits was
determined using the method reported by Bradford, (1976) using bovine serum
albumin as an internal standard.
The data were subjected to analysis of variances. The storage temperatures,
time, varieties, and maturity stages are effect on chilling injury and water and lipids-
soluble antioxidants. The interaction was assessed within the analysis of variances.
The comparisons of difference means were undertaken using the least significant
differences (L.S.D.) at p=0.05. Data was analyzed two periods: the first is from 0 -
20 days, when treatments are presented and the second is from day 0 up to 35
days for all treatment except for treatment at 20oC. Linear regression analysis and
ANOVA were analyzed at the 5% probability level. The statistical software package
Genstat 8 (Lawes Agricultural Trust, Rothamsted Experimental Station, UK) was
used.
88
3. Results
3.1. ASC
Figure 1 shows the changes of ASC (mg per 100 g-1 FW) content in both
mango varieties harvested at three different maturity stages as a function of
storage time (days) at different storage temperatures (2, 4, 7, 10 and 20oC), in the
peel (a) and the pulp (b). In fact, ASC shows significant interactions (p<0.001),
when the storage factors such as time, temperatures, varieties, and maturity stages
were considered. At day zero Zibdia fruits have a three-fold higher ASC content in
both the peel and pulp than Hindi. Zibdia has slightly more ASC in the pulp
compared to the peel at day zero for all maturity stages, whereas in Hindi fruits at
day zero the peel and pulp have similar amounts of ASC The ASC content in the
peel decreases as the fruit maturity increases, though less so for Hindi than Zibdia.
In the pulp of Zibdia fruits the ASC concentration at day zero is relatively unaffected
by maturity stage, whereas in Hindi pulp ASC concentration decreases slightly as
the fruit maturity increases. The ASC content decreases with increasing storage
time for all storage temperatures and maturity stages. The decrease in ASC is
greatest in the pulp of the fruits and is more pronounced in Zibdia than in Hindi
fruits. At 20°C storage the loss of ASC from the pulp was rapid from both Hindi and
Zibdia fruits, so that after 20 days of storage (by which time the fruits had
completely ripened) the level in Hindi had decreased to close to zero for all maturity
stages and in Zibdia the amount ranged from 30 mg 100 g-1 FW at M1 to 10 mg
100 g-1 FW at M3. In the peel at 20°C the loss of ASC was more gradual than in the
pulp so that even though the starting value was lower than for the pulp, after 20
days of storage there was always more ASC in the peel compared to the pulp. The
decrease in ASC during storage at low temperatures was relatively independent of
storage temperature in Hindi fruit. In the peel the decrease is generally least for
fruits stored at 7°C, and in the pulp is least for fruit stored at 10°C. These
differences are, however, small and not significant. In the case of Zibdia the effect
of storage temperature at low storage temperatures is more marked. In both peel
and pulp and for all maturity stages 10°C storage produces the smallest decrease
in ASC content, followed by 7°C, 2°C and finally 4°C. In the case of the peel, the
effect of temperature on ASC content under low temperature conditions was strong.
In comparison to storage at 20°C, storage at low temperatures resulted in a
reduced loss of ASC except for Zibdia peel at the M1 and M2 stages were the
decrease at 20°C was comparable to that at 4°C.
89
Chapter 4
PEEL
120 ZIBDIA HINDI
M1 M1
100
80
60
40
20
Ascorbic acid content of fruit peel (mg 100 g-1 FW)
0
120
M2 M2
100
80
60
40
20
0
120
M3 M3
100
80
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)

Figure 1a. Ascorbic acid content of Zibdia and Hindi varieties harvested in different maturity stages:
immature (M1), half-mature (M2) and full mature fruits (M3), measured of fruit peel versus storage
time at 5 different storage temperatures. Symbols represent the storage temperatures are (Zibdia: -
{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -«-200C).
Ascorbic acid content expressed as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05 for
two period (0- up to 20 days) when all treatments were present and the second is from (0- up to 35
days) for all treatments except for storage at 20oC.
90
PULP
ZIBDIA HINDI
140
M1 M1
120
100
80
60
40
20
Ascorbic acid content of fruit pulp (mg 100 g-1 FW)
0
140
M2 M2
120
100
80
60
40
20
0
140
M3 M3
120
100
80
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 1b. Ascorbic acid content of Zibdia and Hindi varieties harvested in different maturity stages:
immature (M1), half-mature (M2) and full mature fruits (M3), measured of fruit pulp versus storage
time. Symbols represent the storage temperatures are (Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«-
200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -«-200C). Ascorbic acid content expressed as
mean (n=3). The vertical bars indicated to L.S.D. at p=0.05 for two period (0- up to 20 days) when
all treatments were present and the second is from (0- up to 35 days) for all treatments except for
storage at 20oC.
91
Chapter 4
3.2. α-TOC
Figure 2 shows a significant interaction (p<0.001) of α-TOC (mg per 100 g-1
DW) plotted as a function of storage time (days) for both mango fruits harvested at
three maturity stages studied at different storage temperatures (2, 4, 7, 10 and
20oC). Compared to Zibdia a higher α-TOC content was found in Hindi at time of
harvest for most maturity stages, especially in peel. Only in pulp of the M1 stage
did Hindi have a lower α-TOC than did Zibdia. For instance, in Hindi peel α-TOC
was found to range from 15-18 mg per 100 g-1 DW, whereas, it was 9-11 mg per
100 g-1
DW in Zibdia peel. The α-TOC content of the peel and especially the pulp
increased with fruit maturity at day zero for Hindi, whereas for Zibdia fruit maturity
had only a minor effect on α-TOC content. The peel always contained more α-TOC
than the pulp. At all the storage temperatures, α-TOC content decreased during
storage in both mango varieties and for all maturity stages in the peel as well as in
the pulp. Storage at 20oC produced the highest decay rates compared to other
storage temperatures for both varieties at all the maturity stages. At this
temperature there was a strong effect of maturity stage on the loss of α-TOC in
both varieties: in M1 fruits the α-TOC level has decreased to almost zero by 20
days (the end of the storage period at this temperature), whereas for the M3 fruits
the decrease was less severe (approximately 40% loss). Under low temperature
storage the loss of α-TOC from the peel and pulp in Zibdia was least for fruits
stored at 10°C irrespective of the maturity stage, though for the peel the difference
between the 7°C and 10°C temperatures was minor. The greatest decreases in the
α-TOC content of Zibdia under low temperature storage occurred at either 2°C or
4°C, with 4°C storage generally resulting in the greatest decrease. However these
changes occurring under low temperature storage were less than those produced
by storage at 20oC. For Hindi fruits the story is similar: storage at 10°C resulted in
the smallest decrease in α-TOC, except for M2 and M3 pulp where 7°C storage
resulted in a minor improvement in α-TOC retention compared to 10°C, and 4°C
storage was resulted in either the greatest loss of α-TOC or a loss comparable to
that measured in 2°C.
3.3. β-CAR
The β-CAR content in the peel and the pulp of Zibdia at day zero was found
to be higher than that of Hindi, and the β-CAR content in both varieties increased
with increasing maturity stage. For example, the initial β-CAR content in Zibdia peel
92
were 4.39, 5.98, and 6.98 mg per 100 g-1 DW compared with Hindi peel, which
contained 2.34, 3.75 and 5.15 mg per 100 g-1 DW at M1, M2 and M3, respectively.
In the fruit pulp the β-CAR contents were 6.70, 10.64, and 13.74 mg per 100 g-1
DW in Zibdia whereas in Hindi there were no differences among fruit maturity
stages (approximately 13.5 mg per 100 g-1 DW). The changes in β-CAR during
storage are complicated owing to the continued synthesis of β-CAR that occurred
for a limited period at all temperatures. This produced an increase in β-CAR which
was then followed by a decrease in. For fruits stored at 20°C the maximum β-CAR
content was reached at either 10 days (peel of all maturity stages for both varieties)
or 10 or 15 days (pulp). At low storage temperatures the maximum β-CAR content
in the peel occurred at 15 or 20 days and for the pulp at 15 days. In the case of
Hindi pulp the time course of β-CAR content was relatively independent of maturity
stage or storage temperature. For Hindi peel and the peel and pulp of Zibdia, the
time course of β-CAR content was affected by storage temperature, though not
much by maturity stage. In these tissues greatest levels of β-CAR were achieved
when the fruits were stored at 20°C (except in the pulp of M1 of Zibdia). In low
temperature storage, tissues other than pulp from Hindi developed the greatest
concentrations of β-CAR at 10°C and the lowest at 4°C; storage at 2°C resulted in
more β-CAR accumulation, though this sometimes took longer to reach a maximum
value (Hindi peel at all maturity stages, Zibdia peel at M2 and M3 stages). Once the
maximum β-CAR had been reached, the levels declined until the end of the storage
period. In most cases the β-CAR declined to very low levels; sometimes the β-CAR
at after 35 days storage was less than 20% that at the maximum.
3.4. Protein oxidation

Figure 4 depicts the variation of protein carbonyl (PCG) content as a function
of storage time (days) at the three maturity stages for two types of mango fruits
stored at the temperature range (2-20°C) in both the peel (a) and the pulp (b) of the
fruits. In fact, the protein carbonyl content shows a significant interaction at p<0.001
when the storage factors such as time, temperatures, varieties, and maturity stages
were considered. All the maturity stages of both varieties have very low initial levels
(≈zero) protein carbonyl content (PCG) in both the peel and pulp parts of the fruits.
At low storage temperatures the PCG content of the peel part of both varieties
increases more or less linearly with storage time up to about 20 – 25 days of
storage after which a large increase in PCG occurred in some tissue/temperature
accumulations.
93
Chapter 4
PEEL
20 ZIBDIA HINDI
M1 M1
16
12
4
α-Tocopherol content of fruit peel (mg 100 g-1 DW)
0
20
M2 M2
16
12
0
20
M3 M3
16
12
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 2a. α-Tocopherol content of Zibdia and Hindi varieties harvested in different maturity stages:
immature (M1), half-mature (M2) and full mature fruits (M3), measured of fruit peel, versus storage
α-Tocopherol content expressed as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05 for
two period (0- up to 20 days) when all treatments were present and the second is from (0- up to 35
days) for all treatments except for storage at 20oC.
94
PULP
15
ZIBDIA HINDI
M1 M1
12
3
α-Tocopherol content of fruit pulp (mg 100 g-1 DW)
0
15
M2 M2
12
0
15
M3 M3
12
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure2b. α-Tocopherol content of Zibdia and Hindi varieties harvested in different maturity stages:
{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -«-200C). α-
Tocopherol content expressed as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05 for two
period (0- up to 20 days) when all treatments were present and the second is from (0- up to 35 days)
for all treatments except for storage at 20oC.
95
Chapter 4
PEEL
40 ZIBDIA HINDI
M1 M1
30
20
10
β-carotene content of fruit peel (mg 100 g-1 DW)
0
40
M2 M2
30
20
10
0
40
M3 M3
30
20
10
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 3a. β-Carotene content of Zibdia and Hindi varieties harvested in different maturity stages:
immature (M1), half-mature (M2) and full mature fruits (M3), measured of fruit peel versus storage
β-Carotene content expressed as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05 for two
96
PULP
35
ZIBDIA HINDI
M1 M1
30
25
20
15
10
0
β-Carotene content of fruit pulp (mg 100 g-1 DW)
35
M2 M2
30
25
20
15
10
0
35
M3 M3
30
25
20
15
10
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 3b. β-Carotene content of Zibdia and Hindi varieties harvested in different maturity stages:
β-Carotene content expressed as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05 for two
97
Chapter 4
This increase is greatest for older maturity stages and increases as the
temperature is reduced from 10°C to 4°C. Decreasing the temperature from 4°C to
2°C results in reduced increases of PCG to a level intermediate between that
produced by 4°C and 7°C storage. The same behavior can be seen in fruit pulp
samples though the increase in PCG content that develops after 20 – 25 days of
storage is smaller than that found in peel, and in one case (Zibdia M3) the PCG
accumulation at 2°C is greater than that developed at 4°C. At 20°C storage PCG
levels increase rapidly in a comparable way in the peel and pulp of both fruits after
about 5 days of storage. Because of the increase in the peel PCG content that
develops after 20 – 25 days, PCG levels that develop at 20°C are lower than those
the develop in the peel, but much higher than those that develop in the pulp i.e. the
potential for PCG formation is similar in peel and pulp, but the realized PCG
formation under low temperatures is less in the pulp than the peel.
Figure 5 shows the changes of lipid peroxidation, expressed as the

concentration of malondialdehyde equivalent (MDA), in both mango varieties
harvested at three different maturity stages (M1, M2 and M3) as a function of
storage time (days) at different storage temperatures (2, 4, 7, 10 and 20oC) in both
the peel (a) and pulp (b) parts of the same fruits. In fact, the MDA shows a
significant interaction at p<0.001 when the storage factors such as time,
temperatures, varieties, and maturity stages were considered. The initial
concentration is higher in Hindi fruits than in Zibdia fruits in the M2 and M3 stages
owing to an increase in the MDA with maturity in Hindi fruits. In both fruit types,
MDA concentration increases during the storage of fruits, Considering first changes
in fruits stored at low temperatures, in the peel and the pulp either the rate of MDA
accumulation increased as the storage temperature was decreased from 10°C to
4°C or the accumulation was unaffected by temperature in the range 10°C - 4°C.
The only exception to this pattern was found in the peel of Zibdia at for the M2
maturity stage in which storage at 4°C and 10°C produced a similar degree of MDA
accumulation which was higher than that found at 7°C. The behaviour of fruits
stored at 2°C was unusual: for both the peel of Hindi and the pulp of Zibdia at all
maturity stages, and the pulp of Hindi at M1 and M2 stages, there was an increase
of MDA during storage that was greater than that occurring at 4°C. Though this
pattern was also found in Zibdia peel in the M1 stage, in Zibdia peel in the M2 and
M3 stages the increase at 2°C was the lower than for any other temperature.
Storage at 20oC caused a rapid increase in the MDA of the peel of both Zibdia and
98
PEEL
14 ZIBDIA HINDI
M1 M1
12
10
4
Protein carbonyl group content of fruit peel (mM 100 g-1 FW)
0
14
M2 M2
12
10
0
14
M3 M3
12
10
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)

Figure 4a. Protein carbonyl group (PCG) content of Zibdia and Hindi varieties harvested in different
maturity stages: immature (M1), half-mature (M2) and full mature fruits (M3), measured on fruit peel,
temperatures are (Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: - z - 2, - - 4, -▲-
7, -¡- 10 and -«-200C). PCG content expressed as mean (n=3). The vertical bars indicated to
99
Chapter 4
PULP
ZIBDIA HINDI
14
M1 M1
12
10
6
Protein carbonyl group content of fruit pulp (mM 100g-1 FW)
0
14
M2 M2
12
10
0
14
M3 M3
12
10
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 4b. Protein carbonyl group (PCG) content of Zibdia and Hindi varieties harvested in different
maturity stages: immature (M1), half-mature (M2) and full mature fruits (M3), measured on pulps
temperatures are (Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: - - 2, - - 4, -
▲- 7, -¡- 10 and -«-200C). PCG content expressed as mean (n=3). The vertical bars indicated to
100
Hindi to a maximum level after 20 days of storage, and this increase became
greater as the maturity stage at harvest increased. A similar response was found
the pulp of both varieties except that in general the accumulation of MDA was less
than found in the peel.
4. Discussion
One goal of storage is to maintain the levels of nutritionally significant substances,

such as vitamins. In respect of vitamins in mango, only the vitamin content of the
pulp is important; mango peel is not commonly included in processed mango
products, nor is it eaten when the fruit is consumed directly but the condition of the
peel is a fruit quality parameter. So, if changes in antioxidant content can be related
to deterioration of the peel, this would be important in explaining quality changes.
The differences in pulp contents response of ascorbic acid (vitamin C), tocopherol
(vitamin E) and β-carotene (provitamin A) to maturation and storage are important
with respect the optimization of mango as a vitamin source. Zibdia is clearly a much
richer (three-fold) source of ascorbic acid than is Hindi (Ibrahim and Khalif, 1999),
though ascorbic acid levels are not significantly influenced by maturity stage at
harvest. This implies that there is variability, and probably genetic variability, for
ascorbic acid content. This suggests the possibility of selecting or breeding
cultivars with increased ascorbic acid levels. With regards tocopherol in the pulp, a
different pattern of change was identified. For Hindi tocopherol content was strongly
dependent on maturity stage (Barden and Bramlage, 1994), implying that for this
cultivar careful control at the time of harvest is necessary if the tocopherol content
is to maximized. In contrast, in Zibdia the tocopherol content is nearly as high as
the maximum found for Hindi, but it is independent of maturation stage. This
stability is clearly an attractive feature of this cultivar as it removes the need to be
selective at harvest. The β-carotene content of Zibdia and Hindi pulp was similar,
and increased with maturity stage, but given the variability observed in the amounts
of ascorbic acid and tocopherol present at harvest, other cultivars may have
different patterns of β-carotene concentration change during ripening may exist
(Lee and Kader, 2000). These differing patterns of change with maturity stage
present opportunities for both the grower and the breeder to improve the vitamin
content of these fruits. Greater stability of concentration at time of harvest reduces
the need for product selection during the harvesting process, and increased content
at time of harvest would increase the nutritional value of the fruit.
The changes in the concentration of ASC, α-TOC and β-CAR occurring post-
harvest during storage will partly determine the quality of the quality with regards to
the chain. It is evident that the data the post-harvest changes in these compounds
101
Chapter 4
are significant, and that they respond differently to storage; ASC and α-TOC
respond in a similar way, whereas β- CAR behaves quite differently. During storage
the levels of ASC and α-TOC decrease in both the peel and the pulp (Figure 1 and
2). Storage to minimize the loss these antioxidants should be at 10°C, but ideally
the fruit should be used as quickly as possible after harvest. Interestingly, in the
peel ascorbic acid levels decreased less than those in the pulp, so in principle it
should be possible to improve the performance of pulp. In general storage
produced an increase in the β-CAR level during the early period of storage, which
was later followed by a decrease as storage duration increased (Figure 3). The
increase in β-CAR implies that its synthesis continued, and as the increase
occurred even at 2°C this requires that even in this chilling sensitive fruit ordered
metabolism can still occur. It also implies that short-term storage of 10 -15 days
could be used to increase the β-CAR content of the fruit, especially if the fruits are
stored at 10°C or 20°C. For applications where pulp colour is important, such as
juice extraction, this increase in β-CAR level might be valuable (Kader, 2002). As
with other responses, there are differences in the responses of the two cultivars
with regards to their β-carotene responses, which suggests the existence of genetic
variability that could be exploited: the peel of Hindi and the pulp and peel of Zibdia
are quite similar, whereas Hindi pulp has a smaller increase in β-carotene
compared to Hindi peel.
Injury due to exposure of plant tissues to chilling temperatures has been
frequently attributed to oxidative stress (Hodges et al., 2004), and oxidative stress
is a consequence of the balance between the formation of AOS and their
destruction shifting to favour an increased steady-state AOS level (Hodges, 2003).
The formation of both the MDA equivalents and protein carbonyls are due to
oxidative processes (Hodges, 2003), so it would be expected that they might be
correlated. It is important to note that protein carbonyls can be formed as a result of
reactions between proteins and membrane breakdown products (TBARS) as well
as being initiated by hydroxyl ion attack on proteins (Berlett and Stadtman, 1997).
In pulp from both Zibdia and Hindi at all maturity stages under low temperature
storage the correlation is good and similar over the temperature range 2°C - 4°C. At
20°C the correlation is also good and similar for both Zibdia and Hindi, but in this
case the correlation differs substantially from that found at low temperatures: large
increases in PCG occur with only small increases in MDA equivalents (Figures 4
and 5). The relationship between PCG accumulation and the accumulation of MDA
equivalents in peel tissues is much more variable than that found in the pulp,
though it appears to be largely independent of maturity stage. For both Zibdia and
Hindi the responses at 10°C and 20°C are similar.
102
PEEL
ZIBDIA HINDI
2.0
M1 M1
1.6
1.2
0.8
0.4
0.0
2.0
MDA content of fruit peel (ηM g-1 FW)
M2 M2
1.6
1.2
0.8
0.4
0.0
2.0
M3 M3
1.6
1.2
0.8
0.4
0.0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 5a. Malondialdehyde (MDA) content of Zibdia (A) and Hindi (B) cultivars harvested in
different maturity stages: immature (M1), half-mature (M2) and full mature fruits (M3), measured of
fruit peel stored at versus storage time at 5 different storage temperatures. Symbols represent the
- 4, -▲- 7, -¡- 10 and -«-200C). MDA content expressed as mean (n=3). The vertical bars indicated
to L.S.D. at p=0.05 for two period (0- up to 20 days) when all treatments were present and the
second is from (0- up to 35 days) for all treatments except for storage at 20oC.
103
Chapter 4
PULP
ZIBDIA HINDI
2.0
M1 M1
1.6
1.2
0.8
0.4
0.0
MDA content in fruit pulp (ηM g-1 FW)
2.0
M2 M2
1.6
1.2
0.8
0.4
0.0
2.0
M3 M3
1.6
1.2
0.8
0.4
0.0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 5b. Malondialdehyde (MDA) content of Zibdia and Hindi varieties harvested in different
maturity stages: immature (M1), half-mature (M2) and full mature fruits (M3), measured of fruit pulp
stored versus storage time at 5 different storage temperatures. Symbols represent the storage
temperatures are (Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: -z- 2, - - 4, -▲-
7, -¡- 10 and -«-200C). MDA content expressed as mean (n=3). The vertical bars indicated to
104
At lower storage temperatures there are differences between the cultivars: with
decreasing temperatures Hindi has a greater formation of PCG relative to MDA
equivalents, whereas with Zibdia the relative accumulation of PCG and MDA
equivalents is relatively unaffected by temperature.
Taken as a whole these results show that oxidative damage is not a unitary
process. Though the overall trend of oxidation is the same, the degree to which
different cell components can be affected differs. It seems plausible that these
differences are due to either differences in the spectrum of oxidants produced, the
activity of scavenging and repair processes, or both. Protein carbonyls are a
consequence of both hydroxyl radical attack and reactions with membrane
breakdown products, whereas lipid oxidation is more likely to be the product of a
chain reaction involves molecular oxygen, but this process is dependent upon an
initiation event due to an attack by either a hydroxyl radical, or some other radical
(including molecular oxygen) (Baier and Dietz, 1999). In spite of these
complications, the data do show that for specific combinations of tissue,
temperature and cultivar the relationship between the different measures of
oxidation is generally very close, suggesting that though there are can be variations
in the degree to which cell components show net oxidation, there is an underlying
correlation in the oxidation process of these components.
Oxidative stress is a consequence of the balance between the formation of
AOS and their destruction shifting to favour an increased steady-state AOS level
(Foyer and Noctor, 2000). This could be due to either more AOS formation, or less
AOS destruction. The anti-oxidants measured are involved in the elimination of
AOS, but do changes in their concentration correlate with senescence or chilling
related injuries to the fruits, either in the pulp or the peel. It is clear that under low
temperature storage MDA accumulation, an indicator of membrane oxidation
(Figure 4), correlates well with electrolyte leakage (Figure 6), which is an indicator
of membrane damage. The relationship between MDA accumulation and electrolyte
leakage in the pulp is similar for both varieties in the temperature range 4 -10°C. At
2°C and especially at 20°C the increase in electrolyte leakage is relatively more
independent of MDA accumulation. It is possible, however, that at 20°C the rate of
disappearance of MDA from the senescing tissues is comparable to the rate of its
formation, thus masking an underlying correlation between membrane lipid
oxidation and electrolyte leakage. So, membrane damage does seem to correlate
with membrane oxidation, though in some cases this correlation is better than in
others.
In relation to increase of chilling injury index during cold storage, the
changes in antioxidant content in the peel and pulp are revealing. The chilling injury
index is measured by observation of the peel (chapter 3), and thus need not
105
Chapter 4
correlate with processes occurring in the pulp. Nonetheless, as the chilling-injury

index is such a simple means with which assess injury, any correlations that may
exist between peel injury and pulp injury are important to know. At temperatures
where an increase in the chilling-index occurs, all the antioxidants have a broadly
linear correlation between the antioxidant concentration in both pulp and peel and
the CI-index. These relationships were also largely independent of storage
temperature and maturity stage. Zibdia has a much higher ascorbate level in its
peel than Hindi, yet the Zibdia is more chilling sensitive than Hindi and the
increased ascorbate in Zibdia only results in CI-index increasing at higher
ascorbate levels compared to Hindi (Figure 7).
In the case of tocopherol the situation is reversed; the peel of Hindi has
more tocopherol than Zibdia, and this does seem to be associated with a smaller
increase in CI-index in Hindi compared to Zibdia: this is consistent with the greater
chilling tolerance of Hindi compared to Zibdia (Figure 8).
The β-carotene -CI-index correlation is similar for both cultivars. The β-
carotene, concentration of the fruits has a more complicated relationship with
storage-time than the other antioxidant. The increase in β-carotene concentration
precedes the increase in CI-index, and it is the later decrease in β-carotene content
that correlates with the rise in CI-index (Figure 9).
Given the stability of these correlations with temperature and maturity stage
it could be argued that the correlation was more than coincidence and that the
increase in CI-index was mechanistically linked to the decrease in antioxidant
levels in the peel, and less directly to those of the pulp. However, considering only
the peel data, where any mechanistic correlation between antioxidant content and
CI-index would be most direct, a comparison of the relationship between
antioxidant content and CI-index between the two cultivars reveals inconsistencies.
First, at 10°C no increase in CI-index occurs, yet all anti-oxidants undergo changes
in concentration that are as large as those associated with increases in chilling-
injury index at lower temperatures (Chapter 3).
The chilling injury index is based upon an assessment of skin damage
associated with low temperature exposure. The physiology underlying this damage
is not specified, so it is possible that the link with oxidative stress is indirect. Fatty-
acid oxidation, measured by means of the formation of MDA equivalents (Figure 5),
is directly linked to oxidation and thus might be expected to display a better
correlation with antioxidant levels. In the case of ascorbic acid in the peel, the
increase of MDA is well correlated with the loss of ascorbic acid, except at 2°C
where MDA levels remain low even though the ascorbic acid pool decreases
almost to zero (Figure 10).
106
PEEL
2.0
ZIBDIA HINID
o o
2 C y = 0.01x + 0.1136
2 C
y = 0.006x + 0.298
R 2 = 0.8456
1.6 R 2 = 0.8987
y = 0.0053x + 0.3277 y = 0.0049x + 0.3749

1.2 R 2 = 0.8237 R 2 = 0.8487
0.8
0.4 y = 0.0065x + 0.3295 y = 0.0072x + 0.3252

R 2 = 0.6233 R 2 = 0.7957
0.0
2.0
o y = 0.0124x + 0.1006 y = 0.0062x + 0.3774
o
4 C R 2 = 0.8382 4 C R 2 = 0.8652
1.6
y = 0.0152x + 0.1222 y = 0.0097x + 0.3533
1.2 R 2 = 0.9177 R 2 = 0.8884
0.8
0.4 y = 0.0092x + 0.2285 y = 0.0115x + 0.3111

MDA content of fruit peel (ηg 100 g-1 FW)
R 2 = 0.7673 R 2 = 0.9045
0.0
2.0 y = 0.0074x + 0.3698
o y = 0.0061x + 0.2371 o
7 C R 2 = 0.8414 7 C R 2 = 0.7645
1.6
y = 0.0143x + 0.2467
1.2 y = 0.0059x + 0.3122
R 2 = 0.8728
R 2 = 0.7486
0.8
0.4 y = 0.0114x + 0.1768

y = 0.0166x + 0.178
R 2 = 0.9156
R 2 = 0.8356
0.0
2.0
o y = 0.0102x + 0.2786
y = 0.011x+0.1169 o
10 C R2 = 0.7262 10 C R 2 = 0.9161
1.6
y = 0.0122x + 0.2374
y = 0.0169x - 0.0716 R 2 = 0.9506
1.2 R2 = 0.9225
0.8
0.4 y = 0.0194x - 0.0841 y = 0.0211x + 0.0183

R2 = 0.9398 R 2 = 0.921
0.0
2.0 y = 0.007x + 0.3394
o o
20 C 20 C R 2 = 0.8216
1.6 y = 0.014x - 0.0377
R2 = 0.9109 y = 0.0087x + 0.3318
1.2 R 2 = 0.8383
y = 0.0221x - 0.2196
R2 = 0.933
0.8
0.4 y = 0.0249x - 0.289 y = 0.0257x - 0.0936

R2 = 0.9522 R 2 = 0.8065
0.0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 6a. The correlation between lipid peroxidation (MDA) in function α-Tocopherol content of fruit
peel of two mango varieties which stored at different storage temperatures for 35 days, (Zibdia: -{-
2, -- 4, -U- 7, -- 10 and -ª- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -ª- 200C).
Solid lines represent linear regressions.
107
Chapter 4
PULP
2.0
ZIBDIA HINDI
o o y = 0.0086x + 0.2776
y = 0.0063x + 0.2324
2 C 2 C R 2 = 0.9688
1.6 R 2 = 0.863
y = 0.0068x + 0.309
1.2 y = 0.0056x + 0.2142
R 2 = 0.7796
R 2 = 0.6933
0.8
0.4 y = 0.0094x + 0.1969

y = 0.0162x + 0.0938
R 2 = 0.8348 R 2 = 0.9284
0.0
2.0
o o y = 0.016x + 0.0442
4 C y = 0.0124x + 0.1006
4 C R 2 = 0.8348
1.6 R2 = 0.8382
y = 0.0152x + 0.1222 y = 0.0094x + 0.0965
1.2 R 2 = 0.9221
R2 = 0.9177
0.8
0.4 y = 0.0092x + 0.2285

y = 0.0121x + 0.2767
R2 = 0.7673
MDA content of fruit pulp (ηg 100 g-1 FW)
R 2 = 0.8461
0.0
2.0
o y = 0.0338x - 0.8099 o y = 0.0182x - 0.0389
7 C R2 = 0.9036
7 C R 2 = 0.967
1.6 y = 0.0169x - 0.0769
y = 0.0155x - 0.2088 R 2 = 0.8985
1.2 R2 = 0.9279
0.8
0.4
y = 0.0154x + 0.0769 y = 0.0161x + 0.1203
R2 = 0.8381 R 2 = 0.8222
0.0
2.0
o y = 0.0108x + 0.0254 o y = 0.0111x + 0.2304
10 C R 2 = 0.9198
10 C R 2 = 0.8657
1.6 y = 0.011x + 0.062
R 2 = 0.891
1.2 y = 0.0111x - 0.0395
R 2 = 0.7349
0.8
0.4 y = 0.0115x + 0.3982

y = 0.0145x - 0.0931
R 2 = 0.87 R 2 = 0.8504
0.0
2.0
y = 0.0061x + 0.1014
o o y = 0.0061x + 0.3902
20 C R 2 = 0.6746 20 C R 2 = 0.8154
1.6
y = 0.0086x - 0.0882 y = 0.0065x + 0.2897
1.2 R 2 = 0.8203 R 2 = 0.646
0.8
0.4 y = 0.0072x + 0.4297

y = 0.0052x + 0.2471 R 2 = 0.6544
R 2 = 0.6326
0.0
0 20 40 60 80 100 0 20 40 60 80 100
Ion leakage % of fruit pulps

Figure 6b. The correlation between lipid peroxidation (MDA) in function α-Tocopherol content of
fruit pulp of two mango varieties which stored at different storage temperatures for 35 days, (Zibdia:
-{- 2, -- 4, -U- 7, -- 10 and -ª- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -ª- 200C).
Solid lines represent linear regressions.
108
α-Tocopherol levels also show an inverse trend to MDA levels, with the exception
of the 2°C treatment, but the trend is not as clear as for ascorbic acid (Figure 11).
The correlation between β-carotene content and MDA accumulation is, on the other
hand, very poor (Figure 12). With the exception of the 2°C treatment, it seems
therefore, that decreases in ascorbic acid and tocopherol, both of which are
antioxidants that are generally active in the plant cell, are associated with increased
membrane oxidation (Munné-Bosch and Alegre, 2002; Shewfelt and Del Rosario,
2000). Also, the correlations between both tocopherol and ascorbic acid levels, and
MDA equivalent accumulation is similar for both Zibdia and Hindi even though their
contents of these antioxidants differs (Figure 10 and 11). β-carotene, on the other
hand, has antioxidant properties that are important to photosynthesizing tissues,
but otherwise does not appear to an important anti-oxidant in terms of specific
metabolic processes. Notably, MDA accumulation seems to be unrelated to the
loss of β-carotene.
The problem with the link between the loss of active antioxidant and membrane
oxidation is the response at 2°C which shows that antioxidant levels can decrease
without an increase in MDA equivalents. It is possible, however, that low
temperatures are protecting the membrane fatty acids from oxidation. The 4°C
treatments show a greater increase in MDA with the loss of ascorbic acid than does
the 2°C treatments, but it is less than the accumulation found at 7°C. This is
consistent with MDA equivalent formation being inhibited by low temperatures. This
inhibition could be due to various processes ranging from the initial oxidation of the
fatty acid to the breakdown of the oxidized acid to the formation of the MDA
equivalents.
Acknowledgement
The author would like to appreciate the Egyptian High Educational Ministry for
support the PH.D., for full program.
109
Chapter 4
PEEL
120
ZIBDIA HINDI
o y = -20.741x + 113.07 o y = -10.877x + 59.392
2 C 2 C
100 R2 = 0.8749 R2 = 0.7508
80 y = -21.606x + 98.327 y = -10.534x + 54.382

R2 = 0.8577 R2 = 0.7334
60
y = -8.7521x + 41.794
40 R2 = 0.7794
20 y = -15.366x + 72.055
R2 = 0.8785
0
120
o
4 C y = -19.127x + 100.82
4oC y = -8.7964x + 56.56
100 R2 = 0.7563 R2 = 0.8525
y = -8.9933x + 51.824
80 y = -17.582x + 89.321
R2 = 0.863
R2 = 0.7693
60
y = -7.4458x + 41.841
40 R2 = 0.8383
20 y = -13.266x + 67.077
R2 = 0.8428
0
120
7 oC y = -17.189x + 113.93
7oC y = -8.5633x + 60.575
100 R2 = 0.9131
R2 = 0.8889
y = -15.701x + 99.574
80 R2 = 0.8672
y = -9.5535x + 56.619
R2 = 0.8764
60 y = -9.4827x + 45.853
R2 = 0.8575
40
20 y = -12.798x + 75.179
R2 = 0.8648
0
120
10oC 10oC
100
80
60
40
20
0
0 1 2 3 4 50 1 2 3 4 5
Figure 7a. The relationship of ascorbic acid content in function of chilling injury of fruits peel of two
mango varieties stored at different storage temperatures for (2, 4 and 7oC) for 35 days, (Zibdia: -{-
M1, -- M2, and -U- M3) and (Hindi: - z - M1, - z - M2, and -▲- M3). Solid lines represent linear
110
PULP
160 ZIBDIA HINDI
o y = -22.233x + 133.06 o
2 C 2 C y = -7.9855x + 47.695
R2 = 0.8882 R2 = 0.8323
120 y = -24.686x + 129.42
y = -7.3502x + 42.908
R2 = 0.9154
R2 = 0.7296
80
y = -6.123x + 35.562
R2 = 0.6287
40
y = -22.791x + 112.47
R2 = 0.8338
0
160
o
4 C
y = -14.341x + 117.05
R2 = 0.6865
4oC y = -6.8775x + 47.886
120 R2 = 0.8448
Ascorbic acid content of fruit pulp (mg 100 g-1 FW)
y = -19.228x + 111.59
y = -6.648x + 43.232
R2 = 0.7824
80 R2 = 0.851
y = -5.3579x + 34.565
R2 = 0.7332
40
y = -17.313x + 101.49
R2 = 0.7429
0
160
o
7C y = -22.904x + 134.62
7oC y = -8.1137x + 47.319
R2 = 0.8838 R2 = 0.7279
120
y = -31.522x + 132.77
y = -9.2641x + 43.476
R2 = 0.8704
R2 = 0.6508
80
y = -7.0584x + 34.511
R2 = 0.7746
40
y = -26.135x + 110.85
R2 = 0.7329
0
160
10oC 10oC
120
80
40
0
0 1 2 3 4 50 1 2 3 4 5
Figure 7b. The relationship of ascorbic acid content in function of chilling injury of fruit pulp of two
mango stored at different storage temperatures for (2, 4 and 7oC) for 35 days, (Zibdia: -{- M1, --
M2, and -U- M3) and (Hindi: - z - M1, - z - M2, and -▲- M3). Solid lines represent linear
111
Chapter 4
PEEL
ZIBDIA HINDI
20
o y = -3.0774x + 14.289
2oC y = -2.8827x + 11.13 2 C R2 = 0.6403
R2 = 0.8986
15
y = -2.5213x + 15.067
y = -2.5244x + 11.856 R2 = 0.5366
10 R2 = 0.8864
5
y = -2.0807x + 12.471 y = -2.7053x + 16.458
R2 = 0.8906 R2 = 0.6266
α-Tocopherol content of fruit peel (mg 100 g-1 DW)
0
20
4oC y = -2.0024x + 9.004
4oC y = -2.4278x + 13.121
R2 = 0.6396 R2 = 0.7
15 y = -1.9053x + 10.553 y = -2.0737x + 14.856
R2 = 0.7818 R2 = 0.5902
10 y = -1.7459x + 11.536
R2 = 0.8084
5
y = -1.9996x + 16.035
R2 = 0.6638
0
20
o y = -2.3365x + 14.517
7 C y = -1.5811x + 10.826 7oC R2 = 0.6913
R2 = 0.8676
15 y = -2.2719x + 16.523
y = -1.4412x + 11.573 R2 = 0.6771
R2 = 0.8679
10
5
y = -1.3784x + 12.522 y = -2.0578x + 17.436
R2 = 0.7847 R2 = 0.6962
0
20
o o
10 C 10 C
15
10
0
0 1 2 3 4 50 1 2 3 4 5
Chilling injury-index
Figure 8a. The relationship of ascorbic acid content in function of chilling injury of fruit peel of two
mango varieties stored at different storage temperatures (2, 4 and 7oC) for 35 days, (Zibdia: -{-
112
PULP
ZIBDIA HINDI
14
o y = -1.0321x + 5.6071
12
2oC y = -1.9781x + 7.9568
R2 = 0.7824
2 C R2 = 0.419
y = -1.6762x + 9.4879
10 y = -2.0766x + 9.5039
R2 = 0.8218
R2 = 0.8947
8 y = -1.9933x + 11.807
y = -2.0753x + 10.512
R2 = 0.7063
R2 = 0.9069
6
4
2
α-Tocopherol content of fruit pulp (mg 100 g-1 DW)
0
14
4oC y = -1.6855x + 8.5049 4 C
o y = -0.9385x + 6.1404
12 2
R = 0.8622 R2 = 0.8164
10 y = -1.2221x + 8.2626
y = -1.196x + 8.2451
2
R2 = 0.7585
8 R = 0.7806
6
4
y = -1.0313x + 9.3407
2 2
y = -1.4223x + 10.542
R = 0.7451 R2 = 0.7367
0
14
y = -2.6401x + 9.1595 y = -0.9138x + 6.2495
12
7oC 2
R = 0.8682
o
7 C R2 = 0.4959
y = -2.2926x + 9.6005 y = -1.4435x + 9.545
10 2 R2 = 0.7552
R = 0.8415
8 y = -1.7611x + 10.174
2
R = 0.7448
6
4
2 y = -1.3525x + 11.862
R2 = 0.6261
0
14
12 10oC 10oC
10
8
6
4
2
0
0 1 2 3 4 5 0 1 2 3 4 5
Figure 8b. The relationship of ascorbic acid content in function of chilling injury of fruit pulp of two
mango varieties stored at different storage temperatures for (2, 4 and 7oC) for 35 days, (Zibdia: -{-
113
Chapter 4
PEEL
40
ZIBDIA HINDI
o y = -3.6608x + 13.59 o
2 C R2 = 0.7431
2 C y = -3.2734x + 12.386
R2 = 0.5662
30
y = -6.4589x + 23.071
R2 = 0.7338 y = -4.3279x + 15.674
R2 = 0.5762
20 y = -8.4411x + 31.373
R2 = 0.7032 y = -5.0326x + 19.457
R2 = 0.5796
10
0
β-Carotene content of fruit peel (mg 100 g-1 DW)
40
o o y = -2.7329x + 12.914
4 C y = -2.6087x + 10.696 4 C R2 = 0.5336
R2 = 0.521
30 y = -2.642x + 15.175
y = -3.5819x + 14.791 R2 = 0.5031
R2 = 0.6328
20 y = -2.6465x + 17.909
y = -4.2231x + 19.472
R2 = 0.4379
R2 = 0.7156
10
0
40
o y = -5.2508x + 20.265 o y = -2.4404x + 13.28
7 C R2 = 0.5125 7 C
R2 = 0.399
y = -5.3385x + 22.888
30
R2 = 0.5718
y = -2.8236x + 15.877
y = -5.1122x + 26.187 R2 = 0.4483
20 R2 = 0.5135
y = -4.1305x + 21.03
R2 = 0.5132
10
0
40
10 C
o 10oC
30
20
10
0
0 1 2 3 4 5 0 1 2 3 4 5
Figure 9a. presents the relationship of β-carotene content in function of chilling injury of fruit peel of
two mango varieties stored at different storage temperatures (2, 4 and 7oC) for 35 days, (Zibdia: -
{- M1, -- M2, and -U- M3) and (Hindi: - z - M1, - z - M2, and -▲- M3). Solid lines represent
linear regressions on the data.
114
PULP
30
ZIBDIA HINDI
y = -1.1972x + 8.0402 y = -2.1118x + 8.0793
o
2 C R2 = 0.2519 2oC R2 = 0.4424
y = -1.6966x + 11.529
y = -2.5396x + 9.9681
20 R2 = 0.24
R2 = 0.5011
y = -1.7184x + 14.106 y = -1.7184x + 14.106
R2 = 0.2443 R2 = 0.2443
10
0
30
o
4 C
y = -1.7473x + 9.5363
4oC y = -1.0093x + 5.8987
β-Carotene content of fruit pulp (mg 100 g-1 DW)
R2 = 0.5833
R2 = 0.373
20 y = -2.0055x + 11.425 y = -1.2646x + 8.0356

R2 = 0.6482 R2 = 0.3974
y = -2.135x + 13.273 y = -1.4786x + 9.8284

10 R2 = 0.6433 R2 = 0.4718
0
30
o y = -3.3931x + 12.363
7 C R2 = 0.6428
7oC y = -2.5582x + 11.033
R2 = 0.3352
y = -2.1494x + 12.684
y = -3.6612x + 16.057
20 R2 = 0.2225
R2 = 0.5181
y = -2.1105x + 15.362
y = -3.4093x + 17.901
R2 = 0.1798
R2 = 0.4705
10
0
30
10oC 10 C
o
20
10
0
0 1 2 3 4 50 1 2 3 4 5
Figure 9b. presents the relationship of β-carotene content in function of chilling injury of fruit pulp of
two mango varieties stored at different storage temperatures (2, 4 and 7oC) for 35 days, (Zibdia: -
{- M1, -- M2, and -U- M3) and (Hindi: - z - M1, - z - M2, and -▲- M3). Solid lines represent
linear regressions on the data.
115
Chapter 4
PEEL
2.0
ZIBDIA HINDI
y = -0.0051x + 0.835 y = -0.0077x + 0.8374
o o
2 C R 2 = 0.9163 2 C R 2 = 0.9047
1.6
y = -0.005x + 0.8392
y = -0.0066x + 0.7982
R 2 = 0.9052
1.2 R 2 = 0.8948
y = -0.0072x + 0.8918
0.8 R 2 = 0.8556 y = -0.0084x + 0.8047
R 2 = 0.8944
0.4
0.0
2.0
o y = -0.0061x + 0.9684 o
4 C R 2 = 0.9337 4 C y = -0.0144x + 1.1988
R 2 = 0.9734
1.6
y = -0.0109x + 1.2931
y = -0.0183x + 1.322
R 2 = 0.9172
1.2 R 2 = 0.9124
y = -0.0161x + 1.4037
0.8 R 2 = 0.9324 y = -0.0252x + 1.4506
R 2 = 0.8872
0.4
0.0
2.0
o y = -0.0103x + 1.4047 o
7 C R 2 = 0.9439 7 C y = -0.0253x + 1.852
1.6 R 2 = 0.9154
1.2 y = -0.0116x + 1.4361

y = -0.0276x + 1.8835
R 2 = 0.9432
R 2 = 0.9483
0.8
0.4 y = -0.0245x + 2.0349 y = -0.048x + 2.4151

R 2 = 0.9385 R 2 = 0.9555
0.0
2.0
o o y = -0.0213x + 1.5737
10 C y = -0.0242x + 2.7489
10 C R2 = 0.9306
1.6 R2 = 0.8862
1.2 y = -0.0247x + 1.6359

y = -0.0378x + 3.6082 R2 = 0.9397
0.8 R2 = 0.9095
0.4 y = -0.0507x + 3.728 y = -0.0311x + 1.7276

R2 = 0.884 R2 = 0.9495
0.0
2.0
o y = -0.017x + 1.9741 o y = -0.0138x + 1.1793
20 C R 2 = 0.9019 20 C R 2 = 0.9429
1.6
1.2 y = -0.0189x + 2.024

y = -0.0152x + 1.1735
R 2 = 0.9222
R 2 = 0.9239
0.8
0.4 y = -0.0321x + 2.3345 y = -0.0413x + 2.0453

R 2 = 0.807 R 2 = 0.8237
0.0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
Ascorbic acid content of fruits peel (mg 100 g-1 FW)
Figure 10. The relationship of malondialdehyde (MDA) content of fruits peel in function of ascorbic
acid content of fruits peel of two mango varieties were harvested in three different maturity stages
which stored at different storage temperatures for (2, 4, 7 and 10oC) for 35 days, (Zibdia: -{- M1, -
- M2, and -U- M3) and (Hindi: - z - M1, - z - M2, and -▲- M3). Solid lines represent linear
116
PEEL
2.0 ZIBDIA HINDI
y = -0.0361x + 0.6626 o y = -0.0241x + 0.7398
o 2 C
1.6 2 C R 2 = 0.8611 R2 = 0.828
y = -0.0428x + 0.8524
1.2 R 2 = 0.861
y = -0.0237x + 0.8146
R2 = 0.916
0.8 y = -0.0513x + 1.0188
R 2 = 0.779
0.4 y = -0.0251x + 0.8753

R2 = 0.9408
0.0
2.0
y = -0.0542x + 0.856
4 oC R2 = 0.9487
o
4 C
y = -0.0447x + 1.009
R2 = 0.875
1.6
y = -0.1006x + 1.3817
y = -0.0589x + 1.3362
1.2 R2 = 0.9047
R2 = 0.7331
y = -0.1186x + 1.7054
0.8 R2 = 0.9125
0.4 y = -0.0786x + 1.721

R2 = 0.788
0.0
2.0 y = -0.0784x + 1.5138
y = -0.1057x + 1.3935
7oC o
7 C
R2 = 0.889 R2 = 0.8446
1.6
y = -0.1223x + 1.7031
1.2 R2 = 0.8765
0.8 y = -0.2098x + 2.861 y = -0.0987x + 2.0142

R2 = 0.8785 R2 = 0.8857
0.4 y = -0.1922x + 3.6629
R2 = 0.8872
0.0
2.0 y = -0.1412x + 1.6725 y = -0.0832x + 1.668
o o
1.6
10 C R2 = 0.7664 10 C R2 = 0.8355
y = -0.2621x + 3.056 y = -0.0831x + 1.8952
1.2 R2 = 0.8063 R2 = 0.9001
0.8 y = -0.3097x + 3.7342

R2 = 0.8733
0.4 y = -0.112x + 2.5091
R2 = 0.9462
0.0
2.0 o y = -0.0479x + 1.1052
20 C y = -0.169x + 2.2105
o
1.6 R2 = 0.7979 20 C R 2 = 0.8673
y = -0.0844x + 1.0074 y = -0.0578x + 1.3539

1.2 R 2 = 0.9209
R2 = 0.8129
0.8 y = -0.1156x + 1.4707

R2 = 0.8311
0.4 y = -0.1438x + 2.8311
R 2 = 0.8537
0.0
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
α-Tocopherol content of fruit peel mg 100 g-1 DW
Figure 11. The correlation between lipid peroxidation (MDA) in function α-Tocopherol content of
fruit peel of two mango varieties which stored at different storage temperatures for 35 days, (Zibdia:
-{- 2, -- 4, -U- 7, -- 10 and -ª- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -ª- 200C).
Solid lines represent linear regressions on the data.
117
Chapter 4
PEEL
1.8
ZIBDIA HINDI
o y = -0.0193x + 0.5823 o y = -0.0151x + 0.6236
1.5 2 C R 2 = 0.4768 2 C R 2 = 0.4127
y = -0.0087x + 0.6391
1.2 R 2 = 0.2801 y = -0.0083x + 0.6276
R 2 = 0.3077
0.9 y = -0.0063x + 0.6763
R 2 = 0.2451
0.6
y = -0.0064x + 0.648
0.3
R 2 = 0.2285
0.0
1.8
o
4 C y = -0.0257x + 0.7238 o
4 C
y = -0.0211x + 0.7977
1.5 R 2 = 0.445 R 2 = 0.3241
1.2
y = -0.0402x + 1.0111 y = -0.0256x + 0.9626
0.9 R 2 = 0.632 R 2 = 0.2628
0.6
0.3 y = -0.0375x + 1.1694 y = -0.0235x + 1.0789
R 2 = 0.6016 R 2 = 0.1862
0.0
1.8
o y = -0.0133x + 0.6761 o
7 C R 2 = 0.2609 7 C y = -0.0249x + 0.9041
1.5 R 2 = 0.1614
1.2 y = -0.0143x + 0.7817

R 2 = 0.2508 y = -0.0249x + 1.0282

0.9 R 2 = 0.132
0.6
0.3 y = -0.0278x + 1.2131 y = -0.0476x + 1.6405
R 2 = 0.3244 R 2 = 0.2975
0.0
1.8 o y = -0.0292x + 1.01
10 C y = -0.0272x + 0.8697
10 C
o
R 2 = 0.3121
1.5 R 2 = 0.4317
1.2
y = -0.0364x + 1.1065
0.9 R 2 = 0.5595 y = -0.04x + 1.2502
R 2 = 0.418
0.6
0.3 y = -0.0404x + 1.3353 y = -0.0425x + 1.4057
R 2 = 0.5557 R 2 = 0.4496
0.0
1.8
o
20 C y = -0.0158x + 0.7874
o
20 C y = 0.0097x + 0.5524
1.5 R 2 = 0.0357
R2 = 0.1494
1.2
y = 0.0123x + 0.562
y = 0.0071x + 0.6019
0.9 R 2 = 0.04
R2 = 0.0213
0.6
0.3 y = 0.0224x + 0.3838 y = 0.0303x + 0.6026

R 2 = 0.0515
R2 = 0.0864
0.0
0 10 20 30 40 0 10 20 30 40
β-Carotene content of fruits peel (mg 100 g-1 DW)

Figure 12. Presents the relationship of malondialdehyde (MDA) in function of β-Carotene of fruits
peel of two mango varieties were harvest in three maturity stages which stored at different storage
temperatures (2, 4, 7, 10 and 20oC) for 35 days, (Zibdia: -{- M1, -- M2, and -U- M3) and (Hindi:
- z - M1, - z - M2, and -▲- M3). Solid lines represent linear regressions on the data
118
Chapter 5
Antioxidant enzymes and oxidative stress in
mangoes
Loay Arafat1, Jeremy Harbinson1, Olaf van Kooten1, and Linssen JPH2
Horticultural Production Chains Group, Marijkeweg 22, 6709 PG Wageningen
Department of Agrotechnology and Food Sciences, Food Chemistry Laboratories,

Biotechnion, Bomenweg 2, 6703 HD Wageningen University,
The Netherlands
Chapter 5
Abstract
The activities of the antioxidant enzymes superoxide dismutase (SOD), catalase
(CAT), glutathione reductase (GR) and ascorbate peroxidase (APX) were
measured in the peel and pulp of two mango varieties (Mangifera indica L. ‘
Zibdia’ and ‘ Hindi Be-Sennara’) during storage at 4 and 10oC for periods of up to
35 days. Low initial activities of antioxidant enzymes (AE) at harvest time were
observed, though this varied according to the maturity stage of the fruits at harvest.
During the storage period fully mature fruits displayed the highest activities of AE
compared to immature and half-mature fruits. In both varieties AE activities also
increased during the first 15 days of storage, even at the lowest storage
temperatures, although Zibdia showed higher activities of AE activity in this period.
Thereafter, the AE activity decreased during the rest of the storage time. In
conclusion, the AE activity increases up to ≈15 days of storage time after which
they decreased in both the pulp and peel of both varieties and at all maturity
stages. This decrease of AE leads to auto-formation of free radicals. The degree of
oxidative stress pronounced by the cell is a function of the activity of free radical
generation reaction on the one hand, and the activity of the free radicals
scavenging system on the other hand. The imbalance between generation and
scavenging free radicals are attributed to an increased lipid peroxidation, thereafter,
leading to a breakdown of the thylakoid membranes. Chilling injury symptoms are
clearly visible on mango fruits. The storage time and temperature are shown to be
important factors in determining decreases in AE activity, which influence chilling
injury presenting in mango fruits. Decreases in AE activity is varied between the
selected varieties and maturity stages.
Keywords: Mango; superoxide dismutase (SOD); Catalase (CAT); glutathione reductase (GR);
acerbate peroxidase (APX)
120
Antioxidant Enzymes
1. Introduction
Many plants and plant-products of tropical origin suffer injury and even death
when exposed to low but non-freezing temperatures. Chilling sensitive fruits and
vegetables will develop various fairly characteristic symptoms, or disorders, when
stored for sufficient periods of time at temperatures below 15°C, depending on the
species, cultivar, and tissue type. These disorders include: pitting, abnormal
ripping, reduced photosynthetic capacity, necrosis and discoloration and increased
disease susceptibility (Saltveit and Morris, 1990). The causes of this injury are
incompletely understood but seem to involve changes in membrane organization
such as decreases in the unsaturation fatty acids of membrane lipids (Nishida and
Murata, 1996). Additionally, oxidative damage is associated with chilling injury,
which implies that chilling treatments produce a disturbance in the cellular redox
homeostasis of susceptible plant cells (Foyer and Noctor, 2000). In response to
chilling treatments the antioxidant capacity of tissues is frequently observed to
increase, which is consistent with the homeostasis model of Foyer and Noctor
(2000). The formation of active oxygen species (AOS) may be causally related to
alterations in membrane function (Foyer and Noctor, 2003), and AOS are produced
in different microsomal membranes from stressed tissues (Nair et al., 2003).
Therefore, the membrane and AOS models of injury seem to be connected.
The role of oxidative damage produced by AOS, and the degree to which the
cell can maintain its redox balance (Noctor and Foyer, 2003) are important
questions in relation to improved understanding of the processes underlying the
phenomenon of chilling injury. Consistent with the idea of a shift in the oxidative
balance of the cell, it is important to note that AOS are produced normally during
cell metabolism, by enzymic and non-enzymic means; for example, cell membrane
lipids are about 0.6 – 1.7% peroxidised in healthy tissues (Griffiths et al., 2000).
Chilling stress, along with many other stresses, shifts the AOS balance of
the cell and, increases the levels of AOS (Bowler et al., 1992; Foyer and
Harbinson, 1994; Hodges, 2003) and thus the level of oxidative injury experienced
the cell. A range of AOS are produced by metabolic and non-metabolic processes
within the cell (Evans et al., 1999; Shigeoka et al., 2002). In the absence of light the
predominant primary AOS will be superoxide and hydrogen peroxide. Even in
darkness the mitochondrial electron transport system continues to be a source of
O2•-radicals (Bowler et al., 1992), and O2•- is produced by the reduction of O2 by
NADH oxidase in the plasmalemma. From this peroxide can be formed by
superoxide dismutases (Shigeoka et al., 2002), and the peroxide so formed is
destroyed by various mechanisms (Shigeoka et al., 2002), which is caused many
biochemical and molecular changes in cell membrane results in cell death
(Rubinstein, 2000). Though superoxide and peroxide are reactive, they are much
121
Chapter 5
less aggressive than the hydroxyl radical, whose reaction rate constants are close
to diffusion limitation. Though the hydroxyl radical is not known to be produced
directly from a metabolic process, it can be formed via the Haber-Weiss reaction
between superoxide and peroxides in the presence of certain metal ions (e.g. Fe2+).
Once superoxide is formed, peroxide and hydroxyl radicals will form as a result of
SOD activity and the Haber-Weiss reaction.
Various cell components are vulnerable to oxidation by various AOS. The
unsaturated centres of the acyl chains of membrane lipids are particularly
vulnerable to peroxidative chain reactions begun by an initial free-radical attack
(Rubinstein, 2000). Membrane lipid peroxidation reactions make the membrane
less impermeable, more vulnerable to physical damage, and more likely to phase
separate at low temperatures. In addition to attacking membrane lipids, other cell
components, such as lipids, proteins, carbohydrate and, nucleic acid are subject to
attack result in, postharvest disorders such as superficial scald, browning and
loosing of pigments, as reviewed by (Hodges et al., 2004). So, in general, the
electron abstraction reactions produced by free radicals can be characterized as
being indiscriminate and very harmful to living organisms (Abassi et al., 1998)
Superoxide is, however, an notable exception to this rule; though it is a free radical
it is not so aggressive as radicals such as the hydroxyl. This is because of its
negative charge; the protonated superoxide, the hydroperoxyl radical is much
reactive and as a substituent on the acyl chains of fatty acids is an active oxidant in
the free-radical chain reaction. Foyer and Harbinson (1994) clearly indicated that in
all the situations formation/consumption balance of AOS should be tightly
controlled. Therefore, any metabolic imbalances or stress that leads to an increase
in AOS which will cause damage to cell components that may be irremediable. The
continuous oxidative pressure that cells are under results in their having a
comprehensive protection system to guard against injury from AOS. The extent to
which this protective system changes its activity in response to injury attributable to
oxidative processes, or the extent to which an increase in oxidative damage
correlates with decreases in the activity of certain protective mechanisms, are
frequently investigated. Uncovering these correlations is valuable as a priori
evidence of the operation of control systems working to maintain the oxidative
homeostasis of the cell, as well as suggesting which processes may lead to cell
injury and death. Plants have both enzymatic and non-enzymatic antioxidant
systems to prevent or alleviate the damage from AOS. Many enzymes can
efficiently detoxify AOS effects (e.g. lipid hydroperoxidases (Baier and Dietz, 1999)
, whereas others act to detoxify various AOS before they can cause damage, for
example O•-2 is detoxified by SOD (Noctor and Foyer, 1998), and H2O2 is removed
by catalase (CAT) and by reactions catalysed by different kinds of peroxidases e.g.
122
Antioxidant Enzymes
guaiacol (Lepeduš et al., 2004) and glutathione peroxidase (GPX) (Baier and Dietz,
1999). A major H2O2 detoxifying system in plants is the ascorbate-glutathione cycle
that includes APX and GR (Asada, 1994), but this only functions in the chloroplast.
The protection systems are not perfect, however, and an increase in the
production of AOS or a decrease in scavenging capacity will result in an increase in
oxidative damage. Generally, however, plants show a rapid response to increased
AOS produced by physical stresses (in response to, for example, changes in
storage conditions e.g. temperature and time (Rogiers et al., 1998). These changes
lead to an enhancement in cellular oxidants that induce an increase in the activities
of natural antioxidants such as ASC, α-TOC and β-CAR, and antioxidant enzymes
activity such as SOD CAT, GR, and APX (Bowler et al., 1992; Foyer and Harbinson
1994). The effects of storage conditions on the actions of the natural antioxidants
were studied in detail in chapter 3. Saltveit and Morris (1990) have reported that
many fruits present different sensitivities towards chilling temperatures according to
their maturity stages and stage of development. The fruits stages such as ripening,
aging of tissue and duration of exposure to low storage temperature have an
impact on the development of chilling injury. Moreover studies comparing pre and
post-climacteric Avocado, papaya and mango fruits have shown that pre-
climacteric fruits are more sensitive to low storage temperatures than post-
climacteric fruits (Robert, 1990).
In previous chapters damage to mango fruits produced by low temperature
storage were described. These damage processes included oxidation membrane
oxidation, protein oxidation and ion leakage. The changes in the anti-oxidative
metabolites occurring during storage was also reported The aim of this chapter is to
study the changes in the extracted activities of antioxidant enzymes such as CAT,
APX, GR, and SOD during storage of two mango varieties at low temperatures.,
and to try and relate these changes to changes in the anti-oxidative metabolites
and oxidative damage described previously. In other chapters chilling storage
temperatures of2°, 4°, 7° and10°C were used. In this chapter only temperatures if
4° and 10°C were used because of the more complex assays required to measure
enzyme activities; 4°C was used as it frequently produces the most damage and
10°C as it produces less, or even no, stress, depending on the property or process
being measured.
2. Material and methods

2.1. Fruits harvesting and storage condition:
Fruits harvest and transport procedures and schedules are as described in
chapter 3
123
Chapter 5
2.2. Storage setup

Fruits were divided into two batches; each one was composed of 360 Hindi
and 360 Zibdia (120 fruits at each maturity stage in three replicates). These
batches were stored at 4 and 10oC, and ten fruits were taken from each batch per
maturity stage every 5 days (up to 35 days). From these fruits, samples of peel and
pulp were taken and stored at -80oC prior to analysis of ascorbic acid, SOD, CAT,
GR and APX.
2.3. Antioxidant enzymes extraction and assay:

The antioxidant enzymes were extracted using to the method of
(Kingston-Smith et al., 1999). Frozen mango samples (5 g) stored at – 80oC were
crushed into fine powder with a frozen buffer in a ball mill and the pestle under
liquid nitrogen. The soluble proteins were extracted by homogenizing the powder in
5 ml of buffer consisting of 0.1 M sodium phosphate (pH 7.5), 1 mM
ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), 5 mM
ascorbate, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 1 mM
phenylmethylsulfonylfluoride (PMSF), 1 mM p-aminobenzamidine, and 10 µM
leupeptin. The homogenate was centrifuged at 8000g for 5 min at 4oC and the
supernatant was three times as replicates used for the following enzyme assays.
SOD (EC 1.15.1.1.) activity was assayed by monitoring the inhibition of
photochemical reduction of nitro blue terazolium (NBT) based on the method
described by (Giannopolitis and Ries, 1977). Three ml of the reaction mixture
contained 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 µl
NBT, 2 µl riboflavin, 0.1 mM EDTA, and 100 µl of extract. The reaction mixtures
were illuminated for 15 min using a fluorescent light. One unit of SOD activity was
defined as the amount of enzyme required to cause 50% inhibition of the reduction
of the NBT monitored at 560 nm.
CAT (EC 1.11.1.6) activity was determined by following the consumption of
H2O2 (extinction coefficient 39.4 mM-1 cm-1) at 240 nm for 3 min (Aebi, 1984). The
reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 10 mM
H2O2 and 100 µl of extract in a 3 ml volume. The units for CAT activity were µmol
H2O2 min-1 g FW, but “µmol min-1 g FW” will be used in the text.
GR (EC 1.6.4.2) activity was assayed by measuring the oxidation of NADPH
at 340 nm (extinction coefficient 6.2 mM-1 cm-1) for 3 min in 1 ml assay mixture
containing 50 mM potassium phosphate buffer (pH 7.8), 2 mM Na2-NADPH, 50 mM
GSSG and 200 µl of extract The reaction was started by adding NADPH.
Corrections were made for the background of absorbance at 340 nm, without
124
Antioxidant Enzymes
NADPH (Schaedle and Bassham, 1977). The units for GR activity were µmol
NADPH min-1 g FW, but “µmol min-1 g FW” will be used in the text.
APX (EC 1.11.1.11) activity was measured by following the decrease in the
absorbance at 290nm (extinction coefficient 8.2 mM-1 cm-1) for 1 min in 1 ml of
reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM
ascorbic acid, 0.1 mM H2O2 and 200 µl of extract. The reaction was started by
adding enzyme extracts. Correction was made for the low, non-enzymatic oxidation
of ascorbic acid by H2O2 (Nakano and Asada, 1981). The units for APX activity
were µmol ascorbate min-1 g FW, but “µmol min-1 g FW” will be used in the text.
2.4 Ascorbic acid, Malondialdehyde equivalents and protein carbonyl content.

These were measured as described in chapter 4; the amounts of
thiobarbituric acid reactive substances (TBARS) are expressed as
malondialdehyde (MDA) equivalents.

The data were subjected to analysis of variances (ANOVA) with four factors
for temperature, varieties, harvest maturity stage and storage time (days) Means
were compared using the least significantly differences (L.S.D.) at p=0.05. The
comparison of difference means undertaken using the least significant differences
were tested at 5% probability. Linear regression analysis and ANOVA were
analyzed at the 5% probability level. The statistical software package Genstat 8
(Lawes Agricultural Trust, Rothamsted Experimental Station, UK) was used
3. Results
3.1. SOD activity
Figure 1 shows the variation of the superoxide dismutase (SOD) activity
(SOD activity: unit g-1 FW) as a function of storage time (days) for both mango
varieties at two different storage temperatures (4 and 10oC) in the peel (a) and in
pulp (b) parts of Zibdia and Hindi mango varieties harvested at three different
maturity stages (M1, M2 and M3). Clearly, the SOD activity presents a significant
interaction at p<0.001 when the storage factors (time (measured in days),
temperatures, varieties, and maturity stages) were considered. Generally, the peel
and pulp regions of both mangoes types have the same initial SOD activity at
harvest time, though the amount of SOD activity found did change with harvest
stage at maturity. In Zibdia the SOD activity increased from 12 to 30 units g-1 FW)
125
Chapter 5
in both pulp and peel as maturity increased from M1 to M3, whereas in Hindi fruits
the increase was smaller, extending from 20 to 28 units g-1 FW.
PEEL
45 ZIBDIA HINDI
o o
4 C 4 C
30
SOD activity of fruit peel (unit g-1 FW)
15
0
45
10oC 10oC
30
15
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure1a. Superoxide dismutase (SOD) activity of peel of Zibdia and Hindi varieties harvested in
different maturity stages verses storage time: immature (M1), half-mature (M2), and full mature fruits
(M3). Fruits stored at 4 and 10oC for 35 days. Enzymes Symbols represent the different maturity
stages are (Zibdia: -{- M1, -- M2 and -U- M3) and (Hindi: - z - M1, - - M2, and -▲- M3). SOD
activity expressed as mean (n=3 Replicates). The vertical bars indicated to L.S.D. at p=0.05.
After harvest, SOD activity shows an initial period of increasing activity, reaching a
maximum level at day 15 of storage for both varieties at 4 and 10oC. The value of
the maximum activity of SOD increased slightly with increasing maturity stage, and
was not conspicuously influenced by storage temperature, tissue type or cultivar.
The maximum of SOD activity is thereafter decreases gradually till the end of
storage period. The decay rates in SOD activity are more rapid at 4oC than 10oC in
126
Antioxidant Enzymes
both and peel and pulp of both mango varieties. Likewise the decline is greatest for
M3 fruits, then M2 fruits, with M1 fruits showing the slowest decline.
PULP
45 ZIBDIA HINDI
4oC 4oC
30
SOD activity in pulp (unit g-1 FW)
15
0
45
10oC 10oC
30
15
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 1b. Superoxide dismutase (SOD) activity of pulp of Zibdia and Hindi varieties harvested in
different maturity stages verses storage time: immature (M1), half-mature (M2), and full mature fruits
(M3). Fruits stored at 4 and 10oC for 35 days. Symbols represent the different maturity stages are
(Zibdia: -{- M1, -- M2 and -U- M3) and (Hindi: - z - M1, - - M2, and -▲- M3). SOD activity
expressed as mean (n=3 Replicates). The vertical bars indicated to L.S.D. at p=0.05.
3.2. CAT activity
Figure 2 shows the variation of catalase (CAT) activity (µmol min-1 g FW) as
a function of storage time (days) for both mango varieties at two different storage
temperatures (4 and 10oC) in the peel (a) and pulp (b) of Zibdia and Hindi mango
varieties harvested at three different maturity stages.
127
Chapter 5
PEEL
ZIBDIA HINDI
40
o o
4 C 4 C
35
30
25
20
15
10
CAT activity of fruit peel (µM g-1 FW)
0
40
o
10 C o
10 C
35
30
25
20
15
10
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 2a. Catalase (CAT) activity of peel of Zibdia and Hindi varieties harvested in different
maturity stages verses storage time: immature (M1), half-mature (M2) and full mature fruits (M3),
stored at 4 and 10oC for 35 day. Symbols represent the different maturity stages are (Zibdia: -{-
M1, -- M2 and -U- M3) and (Hindi: - z - M1, - - M2, and -▲- M3). CAT activity expressed as
mean (n=3 Replicates). The vertical bars indicated to L.S.D. at p=0.05.
Generally, the CAT activity shows a significant interaction at p<0.001 when the
storage time, temperature, varieties and maturity stages were considered. At the
time of harvest the CAT activity increased with increasing maturity stage (as with
SOD), but comparing varieties or tissue types, the differences were not striking.
However, irrespective of maturity stage, cultivar, tissue type or storage temperature
CAT activity initially increased during storage. In the case of M3 fruits of Hindi this
increase were substantial: in the peel of Zibdia fruits the increase was in the range
23-53%, in the peel of Hindi it was 8-60%. Maximum CAT activities were achieved
after 5 – 15 days of storage after which activity decreased to reach a minimum
value after 25 days of storage or longer. CAT activity following this decrease was
128
Antioxidant Enzymes
either independent of maturity stage or storage temperature, or increased with

increasing maturity stage. Ultimately, CAT activity was lower at the end of the
storage period than it was at the beginning.
PULP
ZIBDIA HINDI
40
o
4 oC 4 C
30
20
CAT activity of fruit pulp (µM g-1 FW)
10
0
40
o o
10 C 10 C
30
20
10
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 2b. Catalase (CAT) activity of pulp of Zibdia and Hindi varieties harvested in different
maturity stages verses storage time: immature (M1), half-mature (M2) and full mature fruits (M3),
M1, -- M2 and -U- M3) and (Hindi: - z - M1, - - M2, and -▲- M3). CAT activity expressed as
mean (n=3). The vertical bars indicated to L.S.D. at p≤0.05.
3.3. GR activity
Figure 3 shows the variation of GR activity (µmol min-1 g FW) as function of

storage time (days), at different storage temperatures (4 and 10oC) in the peel (a)
and pulp (b) for both mango varieties harvested at three different maturity stages.
129
Chapter 5
In fact, the GR activity shows a significant interaction at p=0.01 when the storage
time, temperature, varieties and maturity stages were considered. Generally, the
peel and pulp of Zibdia fruits and the pulp of Hindi fruits had a slightly lower initial
GR activity compared to the peel of Hindi fruits. The initial activities of GR was
independent of maturity stage. Subsequently during storage GR activity increased
reaching a maximum after 10 – 15 days of storage. This increase in activity was up
to about 20-fold, and it varied depending on the tissue type, maturity stage, and
temperature. In general it was greater at 4°C than at 10°C, it was greater with
increased maturity stage, and for the Zibdia the increase was greater in the peel
than the pulp.
PEEL
100
ZIBDIA HINDI
o
4 C o
4 C
80
60
GR activity of fruit peel (µM g-1 FW)
40
20
0
100
o o
10 C 10 C
80
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 3a. Glutathione reductase (GR) activity of peel of Zibdia and Hindi varieties harvested at
different maturity stages verses storage time: immature (M1), half-mature (M2) and full mature fruits
(M3), stored at 4 and 10oC for 35 day. Symbols represent the different maturity stages are (Zibdia: -
{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). GR activity expressed as
mean (n=3). The vertical bars indicated to L.S.D. at p=0.05.
130
Antioxidant Enzymes
PULP
100
ZIBDIA HINDI
o o
4 C 4 C
80
60
GR activity of fruit pulp (µM g-1 FW)
40
20
0
100
o
10 C 10 C
o
80
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 3b. Glutathione reductase (GR) activity of pulp of Zibdia and Hindi varieties harvested at
{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). GR activity expressed as
mean (n=3). The vertical bars indicated to L.S.D. at p=0.05.
The maximum of GR activity is followed by a decrease in activity that

extended to the end of the experiment. In most cases the activity at the end of the
experiment was greater at 4°C than at 10°C, and if there was any difference
between different maturity stages GR activity was greater with increasing maturity
stage. At 4°C the activity at the end of the experiment was always greater than the
initial activity, whereas at 10°C storage final activity was sometimes lower than
initial activity.
131
Chapter 5
3.4. APX activity

Figure 4 displays the variation of APX activity (µmol min-1 g-1 FW) as function
of storage time (days) for both mango varieties at different storage temperatures
(4 and 10oC) in the peel (a) and pulp (b). In fact, the APX activity shows a
significant interaction at p<0.001 when the storage time, temperature, varieties and
maturity stages were considered. It is apparent from Figure 7 that, both mango
types have almost the initial APX activity was independent of tissue type and
cultivar, but increased with increasing maturity stage.
PEEL
ZIBDIA HNIDI
80
o o
4 C 4 C
60
40
APX activity of fruit peel (µM g-1 FW)
20
0
80
o o
10 C 10 C
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 4a. Ascorbate peroxidase (APX) activity of peel of Zibdia and Hindi varieties harvested in
{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). APX activity expressed
as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05.
132
Antioxidant Enzymes
As observed with both CAT and GR, the initial activity of APX increases
during storage to a maximum activity depending on the mango cultivar, the maturity
stage, and the storage temperatures. The activity reaches the maximum after 5 - 15
days of storage. The maximum activity was independent of cultivar and storage
temperature, but increased with increasing maturity stage. With increasing duration
of storage APX activity progressively decreased, and even at the end of the storage
period this decrease had not ended.
PULP
ZIBDIA HINDI
80
o o
4 C 4 C
60
40
APX activity of fruit pulp (µM g-1 FW)
20
0
80
o o
10 C 10 C
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 4b. Ascorbate peroxidase (APX) activity of pulp of Zibdia and Hindi varieties harvested in
{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). APX activity expressed
as mean (n=3). The vertical bars indicated to L.S.D. at p=0.05.
133
Chapter 5
In all cases, however, by the end of the storage treatment APX activities had
decreased to below the initial activities Overall both mango varieties show a
variation of APX activity dependent on fruit maturity stages, i.e. M3>M2>M1 at both
storage temperatures and tissue types.
3.5 Ascorbic acid, Malondialdehyde equivalents and protein carbonyl content.
These results were described in details in chapter 4
4. Discussion
Peroxidation of cell lipid membranes with resulting in AOS formation has
been described previously as one of the most damaging processes for membranes.
Unsaturated fatty acids are known to easily attacked by lipoxygenase and an
increased AOS production has been observed in a variety of plant tissues. The
content of saturated fatty acids in membranes, lipids is strongly suggested to be
directly linked to chilling tolerance in plants. However, unsaturated fatty acids are
easily peroxidized by hydroxyl radicals that are formed from O2• – (Marangoni et al.,
1996), by lipoxygenase et..
The breakdown of peroxidated lipids results in the formation of a multiplicity
of low molecular weight volatile compounds (Cava et al., 2000). Because many of
these compounds have low odour and taste thresholds they are responsible for
flavours producing either positive or negative consumer reactions (Labuza, 1971).
Marangoni et al., (1996) have reported that the extent to which the autooxidation of
fatty acids and esters contributes to the formation of volatile compounds depends
on both the chemical structure of the fatty acid and the process and/or the storage
temperature. In principal, unsaturated fatty acids are much more sensitive than
saturated ones towards autooxidation and only the former become more sensitive
to oxidation under normal storage conditions. The mechanism of lipid peroxidation
has been discussed in Chapter 2. Malondialdehyde (MDA) and other aldehydes
constitute one of the major groups of products formed following lipid peroxidation,
so determination of these compounds as thiobarbituric acid reactive substances is,
therefore, a sign of the degree of lipid peroxidation. Storage of mangoes at 20oC
caused rapid increase in the MDA content during storage (Chapter 4), which
implies membrane oxidation and breakdown. Membrane oxidation alters bilayer
stability and increases the permeability of the membrane (see chapter 4). The
same feature has previously been observed during the membrane breakdown
which occurs during normal ripening (Hodges, 2003; Meir, 1986). More important
for this study are the responses that occur when mango fruits were stored between
4 and 10oC. It is clear that lipid peroxidation (MDA) increases as the storage
134
Antioxidant Enzymes
temperature is decreased, though in a small number of treatments (e.g. M2 and M3

Zibdia peel and M3 Hindi pulp) the greatest damage occurred at 4°C, not 2°C.
These results indicate that low storage temperatures increase lipid peroxidation of
cell membrane, which also correlates with decreases in ascorbic acid and
tocopherol contents, and increases in electrolyte leakage (chapter 4). This is
consistent with the idea that the consequences of the peroxidation process are
losses in cell membrane integrity, physical structure, membrane fluidity, and
increase permeability. Overall, Zibdia fruit pulp accumulates less MDA than does
the pulp of Hindi, but the relationship between MDA accumulation and ascorbic
acid content is similar for both fruit varieties at each storage temperature. This
strengthens the proposition that oxidative damage is occurring, and that the rate of
damage is inversely dependent upon antioxidant capacity as suggested by the
redox homeostasis model of Foyer and Noctor (2000).
In addition to membrane oxidation, oxdatively modified forms of proteins also
accumulate during storage (chapter 4). This is an oxidative stress that is initiated by
and dependent upon the presence of AOS; hydroxyl radicals are thought to initiate
the reaction by forming protein radicals which then react further with O2, O2-., or
HO2., or oxidation may occur by the reaction of proteins with the breakdown
products of lipid peroxidation (Berlett and Stadtmann, 1997). Collectively, these
active oxygen species lead to oxidation of amino acid residue side chains,
formation of protein-protein cross linking, and oxidation of the protein backbone,
and finally, protein fragmentation (Berlett and Stadtman, 1997). The presence of
carbonyl groups on proteins has been used as a marker of AOS-mediated cell
oxidation and as it is relatively easy to detect using simple protocols, so it is a
relatively sensitive, reliable indicator (Levine et al., 1994). It is clear from our data
that as with membrane oxidation the protein oxidation processes depend on
storage temperatures (Chapter 4). The effect of decreased storage temperatures is
to enhance the oxidative reactions, though in contrast to MDA formation, the
greatest oxidation always occurs at 4°C, not 2°C.The relationship between protein
oxidation (PCG) and AE (APX, CAT and SOD), shows that these is an increase in
PCG when AE activities decreased after 15th day of storage (Figure 5, 6 and 7).
Therefore, it seem that the increases of AOS formation during the first 15 days of
storage are parallel to increases of AE activities, thereafter, AE decreased up to 35
days. These relationships were different depend on variety and maturity stage;, the
effects of maturity stage, storage time and temperature have been reviewed by
(Hodges et al., 2004). All four enzymes, SOD, CAT, GR, and APX, show similar
trends during storage.
135
Chapter 5
PEEL
ZIBDIA HINDI
14
y = -0.1072x + 6.288
4o C y = -0.0467x + 3.1689 o
4 C R2 = 0.4473
12 R2 = 0.42
y = -0.1172x + 8.2705
Protein carbonyl content of fruit peel (mM100 g-1 FW)
10 y = -0.0584x + 4.4035
R2 = 0.3403
R2 = 0.5223
8
y = -0.0555x + 4.8809 y = -0.1047x + 8.6708
6 R2 = 0.4164 R2 = 0.2985
0
14
o y = -0.1724x + 7.7223 o y = -0.2364x + 10.185
10 C R2 = 0.5094 10 C R2 = 0.5931
12
y = -0.197x + 12.765
y = -0.1828x + 10.301
10 R2 = 0.6892
R2 = 0.4473
8 y = -0.2509x + 16.673 y = -0.1952x + 14.266

R2 = 0.8001 R2 = 0.3048
6
0
0 20 40 60 80 0 20 40 60 80
Figure 5a. The relationship protein carbonyl (PCG) content in fruits peel in function of ascorbate
peroxidase activity in fruits peel of two mango varieties Zibdia and Hindi were harvested in different
maturity stages were stored at 4 and 10oC for 35 days. Symbols represent the different maturity
stages are (Zibdia: -{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). Solid
lines represent linear regression.
They all show some degree of increase during the storage period, and
subsequently a decline in activity. Where activity is dependent on maturity stage it
increases with increasing maturity stage, and where activity depends upon tissue
type, the fluctuations in activity are more dramatic in the peel compared to the pulp.
Surprisingly, the increases in enzyme activity occur even at 4°C, and in some
cases the increases are greatest at this temperature. This implies that though this
is a chilling sensitive tropical fruit, it is still capable of protein biosynthesis, which
implies a high level of metabolic control and coordination. Notably, β-carotene
content also increases during the cold storage of mangoes (Kader, 2002) chapter
4, reaching a maximum after 15 days at 4 ° C, and 10°C. This increase also implies
coordinated metabolic activity. Increases in chilling-injury index and, less clearly ion
leakage, occur after 15 days of cold storage (Chapter 3), so the maximum of
136
Antioxidant Enzymes
enzyme activity may be determined by the onset of cell damage induced by the
cold-storage.
PULP
ZIBDIA HINDI
8
o y = -0.054x + 3.292 y = -0.1362x + 6.8355
4 C R2 = 0.3267 4oC 2
R = 0.7539
7
y = -0.1127x + 7.4226
y = -0.0621x + 4.5027 2
6 R = 0.4712
R2 = 0.5077
Protein carbonyl content of fruit pulp (mM100 g-1 FW)
5 y = -0.0703x + 6.1733 y = -0.1018x + 7.8028

2
R2 = 0.3654 R = 0.4609
4
0
8
o y = -0.039x + 2.7969 o y = -0.0337x + 2.743
7
10 C R2 = 0.3757
10 C
R2 = 0.302
6 y = -0.0477x + 3.5799
y = -0.0219x + 2.7822
R2 = 0.4721
R2 = 0.0681
5
y = -0.0427x + 3.8382
R2 = 0.3073 y = -0.0287x + 3.4271
4
R2 = 0.1155
0
0 20 40 60 80 0 20 40 60 80
APX activity of fruits pulp (µM g-1 FW)
Figure 5b. The relationship protein carbonyl (PCG) content in fruits pulp in function of ascorbate
peroxidase activity (APX) in fruits pulp of two mango varieties Zibdia and Hindi were harvested in
different maturity stages were stored at 4 and 10oC for 35 days. Symbols represent the different
maturity stages are (Zibdia: -{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲-
M3). Solid lines represent linear regression.
The responses of anti-oxidant enzymes are different to those of the changes in the
pool size of anti-oxidant metabolites (ascorbic acid α-tocopherol and β-carotene).
Both ascorbic acid and tocopherol show continuous decreases in pool size with
increasing duration of storage. Only the β-carotene pool displays the increase in
size found with the enzymes.
137
Chapter 5
PEEL
ZIBDIA HINDI
7
4o C
o y = -0.1598x + 3.7745 y = -0.3154x + 6.5229
4 C R2 = 0.7179
6 R2 = 0.6509
y = -0.1426x + 4.1182 y = -0.3315x + 8.1705

5 R2 = 0.6066 R2 = 0.7689
4 y = -0.1452x + 5.1171 y = -0.1524x + 6.0964

Protein carbonyl content of fruit peel (mM100 g-1 FW)
R2 = 0.6112 R2 = 0.4497
3
0
14
o y = -0.1977x + 3.8314 o
10 C R2 = 0.2071 10 C y = -0.3927x + 8.5706
2
12 R = 0.6841
y = -0.2619x + 5.9208 y = -0.5314x + 12.446

10 R2 = 0.3962 2
R = 0.7793
8 y = -0.4921x + 13.314
2
y = -0.2827x + 8.2329 R = 0.6621
R2 = 0.4228
6
0
0 10 20 30 40 0 10 20 30 40
Figure 6a. presents the relationship protein carbonyl (PCG) content in fruits peel in function of
catalase activity (CAT) in fruits pulp of two mango varieties Zibdia and Hindi were harvested in
different maturity stages were stored at 4 and 10oC for 35 days. Symbols represent the different
maturity stages are (Zibdia: -{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲-
M3). Solid lines represent linear regression.
138
Antioxidant Enzymes
PULP
ZIBDIA HINDI
8
o y = -0.2248x + 4.3253 o y = -0.3067x + 5.3709
4 C 4 C
7 R2 = 0.8225 R2 = 0.8644
6 y = -0.2348x + 5.8286 y = -0.2514x + 5.4334

R2 = 0.4374 R2 = 0.7574
5
y = -0.1405x + 4.8154
y = -0.3012x + 8.6625
4 R2 = 0.5627
Protein carbonyl content of fruit pulp (mM100 g-1 FW)
R2 = 0.728
0
4
o y = -0.1367x + 2.638 o y = -0.1329x + 2.6627
10 C R2 = 0.6787
10 C
R2 = 0.8012
y = -0.1251x + 2.8022 y = -0.1197x + 3.7566

3
R2 = 0.6859 R2 = 0.5545
y = -0.096x + 3.1282
R2 = 0.7975 y = -0.101x + 4.0125
2
R2 = 0.4131
0
0 10 20 30 400 10 20 30 40
Figure 6b The relationship protein carbonyl (PCG) content in fruits pulp in function of catalase
activity (CAT) in fruits pulp of two mango varieties Zibdia and Hindi were harvested in different
This is in contrast to changes in anti-oxidant pool sizes and enzyme activities found
in other tissues where changes typically occur in parallel. To explore to what extent
the changes in anti-oxidant enzyme activity correlates with cell injury the
relationship between anti-oxidant enzyme activity and chilling injury and ascorbic
acid levels was further explored (Figure 8 and 9). The production of thiobarbituric
acid reactive substances (TBARS), expressed as malondialdehyde equivalents
(MDA equivalents) increases during the cold storage of mangoes (Chapter 3) due
to the oxidation of unsaturated acyl groups of membrane lipids.
139
Chapter 5
PEEL
ZIBDIA HINDI
7
o
4 C
y = -0.0661x + 3.162 4 oC
6 R2 = 0.2561
y = -0.1954x + 7.2828
5 y = -0.0864x + 4.2413
R2 = 0.4522
Protein carbonyle content of fruit peel (mM100 g-1 FW)
R2 = 0.1707
3 y = -0.2253x + 8.9782
R2 = 0.2475
2
y = -0.1469x + 6.799
1 y = -0.2622x + 10.875
R2 = 0.1264
R2 = 0.243
0
14
y = -0.134x + 5.0877
10oC 10 C
o
12 R2 = 0.0588 y = -0.42x + 13.534
R2 = 0.3122
10
y = -0.2598x + 9.6895 y = -0.336x + 13.09
8 R2 = 0.1217 R2 = 0.2276
6
y = -0.2607x + 12.35
4 R2 = 0.0617
y = -0.9511x + 33.771
R2 = 0.5705
2
0
0 10 20 30 40 0 10 20 30 40
SOD activity in fruits peel (unit g-1 FW)
Figure 7a. The relationship protein carbonyl (PCG) content in fruits peel in function of superoxide
dismutase (SOD) in fruits peel of two mango varieties Zibdia and Hindi were harvested in different
The accumulation of MDA equivalents correlates with the increase in electrolyte

leakage that develops during cold storage (Chapter 3). Using data for the
accumulation of MDA equivalents reported elsewhere (chapter 4), the relationship
between membrane oxidation, and the activity of anti-oxidant enzymes can be
explored (Figure 10, 11, 12 and 13). It is evident that APX activity is in broad terms
inversely correlated with the accumulation of MDA equivalents (Figure 10).
140
Antioxidant Enzymes
PULP
ZIBDIA HINDI
8
y = -0.0152x + 1.9298 4oC y = -0.0882x + 4.4544 o

4 C
7 R2 = 0.1886
R2 = 0.0177
6
y = -0.0988x + 5.1887
Protein carbonyle content of fruit pulp (mM100 g-1 FW)
5 R2 = 0.1151
y = -0.0422x + 3.1514
4 R2 = 0.0412
2
y = -0.1069x + 5.8525
y = -0.1083x + 6.0918 R2 = 0.0643
1
R2 = 0.1087
0
4
y = -0.0471x + 2.4277 10oC y = -0.0635x + 2.8259 o
10 C
2
R = 0.1648 R2 = 0.2099
3
y = -0.0059x + 1.8522
y = -0.0737x + 3.432 R2 = 0.0009
R2 = 0.1998
2
1 y = -0.0526x + 3.264
2
R = 0.0929 y = 0.0303x + 1.0138
R2 = 0.0382
0
0 10 20 30 40 0 10 20 30 40
SOD activity of fruit pulp (unit g-1 FW)
Figure 7b. The relationship protein carbonyl (PCG) content in fruits pulp in function of superoxide
dismutase (SOD) in fruits pulp of two mango varieties Zibdia and Hindi were harvested in different
However in detail the correlation is often weak, especially in the pulp. With catalase
activity the correlation with the accumulation of MDA equivalents is clearer,
especially in the peel. Both CAT and APX act to destroy peroxide, though acting by
different mechanisms: CAT acts alone, whereas APX requires ascorbic acid.
141
Chapter 5
PEEL
ZIBDIA HINDI
120
y = 1.7999x - 2.4168 o y = 0.6539x + 13.73 o
2
4 C R2 = 0.3921 4 C
R = 0.7609
100
y = 1.0467x + 7.0898 y = 0.6154x + 2.834
2
R2 = 0.2387
R = 0.3873
80
y = 0.8053x - 3.0469 y = 0.5586x - 4.7616
2
R = 0.3711 R2 = 0.3041
60
40
20
0
120
y = 0.7367x + 63.312 o y = 0.8853x + 11.052 o
10 C R2 = 0.5228
10 C
R2 = 0.7751
100
y = 0.4735x + 10.884
R2 = 0.2428
80
y = 0.4461x + 3.4206
60 R2 = 0.2102
40
y = 0.3865x + 61.573
R2 = 0.4798
20
y = 0.3728x + 40.12
R2 = 0.6385
0
0 20 40 60 80 0 20 40 60 80
Figure 8a. The relationship of ascorbic acid content in function of ascorbate peroxidase (APX)
activity on fruits peel of Zibdia and Hindi varieties harvested in different maturity stages were stored
at 4 and 10oC for 35 day. Symbols represent the different maturity stages are (Zibdia: -{- M1, --
M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). Solid lines represent linear
regression.
Ascorbic acid levels are closely inversely correlated with the accumulation of MDA
equivalents. This could be interpreted as evidence that the protective, anti-oxidant
property of APX is limited by ascorbic acid supply, however it must be borne in
mind that ascorbic acid has varied functions in anti-oxidative metabolism (e.g.
regeneration of oxidized tocopherol) and it may be another function that is the
source of the good correlation between ascorbic acid and the accumulation of MDA
equivalents. The accumulation of MDA equivalents appears to be largely
independent of SOD activity in either the peel or the pulp. This would imply that
changes in SOD activity play no major role in the increase of membrane oxidation.
142
Antioxidant Enzymes
This is not to say that SOD activity is not important: SOD is a required for aerobic
life, but it may be that the oxidative stress leading to membrane damage has not
relation to superoxide formation, or it may mean that SOD was present in excess. It
could also be that a specific form of SOD in a specific cell-compartment was would
have a correlation with MDA equivalent formation.
PULP
140
ZIBDIA HINDI
y = 1.0144x + 56.501 o o
4 C y = 0.818x + 5.6264
4 C
120 R2 = 0.29 R2 = 0.7606
100 y = 1.2873x + 18.516

Ascorbic acid content in pulp (mg 100 g-1 FW)
R2 = 0.5385 y = 0.6382x + 0.5657

80 R2 = 0.5137
60
y = 0.4095x + 2.381
40 R2 = 0.3692
20 y = 0.88x + 20.551
R2 = 0.2775
0
140
y = 0.9332x + 50.722 o y = 0.5331x + 13.574 o
10 C 10 C
R2 = 0.3026 R2 = 0.5182
120
100
y = 1.504x + 7.7032 y = 0.619x + 0.3968
R2 = 0.5019 R2 = 0.3635
80
60
y = 0.492x - 4.1654
40 R2 = 0.4673
20 y = 1.4179x - 4.0106
R2 = 0.3807
0
0 20 40 60 80 0 20 40 60 80
APX activity in pulp (µM g-1 FW)
Figure 8b. Presents the relationship of ascorbic acid content in function of ascorbate peroxidase
(APX) activity on fruits pulp of Zibdia and Hindi varieties harvested in different maturity stages were
M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). Solid lines represent linear
regression.
As with SOD, GR activity likewise has no correlation with the accumulation of MDA
equivalents, and as with the SOD this may imply that the oxidative syndrome
143
Chapter 5
leading to oxidation of fatty acids is not affected by GR activity, or that GR activity

was saturating.
PEEL
ZIBDIA HINDI
120
o y = 3.816x + 10.17 o y = 2.051x + 10.54
4 C 2 4 C
R = 0.5857 R2 = 0.7157
100
y = 2.6211x + 11.104
2 y = 2.0292x - 1.5807
R = 0.4737
80 R2 = 0.7333
60 y = 0.838x + 8.4445
R2 = 0.4864
40
20 y = 2.1643x - 7.739
2
R = 0.5755
0
120
o y = 1.3254x + 74.641 o y = 1.7633x + 12.866
10 C R2 = 0.7753
10 C
R2 = 0.8667
100
y = 1.9018x + 1.1604
80
R2 = 0.9384
60
y = 0.9103x + 65.996
40 R2 = 0.745
20 y = 1.5231x - 2.0392
y = 0.5539x + 50.558
R2 = 0.587 R2 = 0.8374
0
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35
Figure 9a.Presents the relationship of ascorbic acid content in function of catalase (CAT) activity on
fruits peel of Zibdia and Hindi varieties harvested in different maturity stages were stored at 4 and
10oC for 35 day. Symbols represent the different maturity stages are (Zibdia: -{- M1, -- M2 and -
U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). Solid lines represent linear regression.
It may also be that as with APX and ascorbate, the levels of glutathione might limit
the anti-oxidative activity of GR. It has earlier been shown that protein carbonyl
formation is closely correlated with the accumulation of MDA equivalents. This
being so, it is evident that similar correlations would be expected between protein
carbonyl formation and anti-oxidative enzyme activity. In both the peel and the pulp
the activity of CAT has a good inverse correlation with protein carbonyl formation
144
Antioxidant Enzymes
(Figure 6), whereas with APX this correlation is weaker, and with SOD it is very
poor (Figure 7).
PULP
ZIBDIA HINDI
120
o o y = 1.8257x + 14.593
4 C y = -1.0061x + 86.481 4 C
2
R = 0.1865 R2 = 0.8569
100
y = 1.343x + 12.836
80
Ascorbic acid content in pulp (mg 100 g-1 FW)
R2 = 0.7342
y = -0.6577x + 75.545
2
R = 0.2174
60 y = 0.6125x + 13.636
R2 = 0.5301
40
20
y = -0.3061x + 51.886
2
R = 0.1303
0
120
o y = 1.5277x + 20.368
10oC y = 0.086x + 87.722 10 C R2 = 0.7253
R2 = 0.0157
100
y = 1.6534x + 2.5404
80 R2 = 0.7089
60 y = 1.0755x - 0.1043
R2 = 0.6472
y = -0.0228x + 78.891
40 R2 = 0.0018
20 y = 0.0413x + 58.16
R2 = 0.0081
0
0 20 40 60 0 10 20 30 40
CAT activity in pulp (µM g-1 FW)
Figure 9b. The relationship of ascorbic acid content in function of catalase (CAT) activity on fruits
pulp of Zibdia and Hindi varieties harvested in different maturity stages were stored at 4 and 10oC
for 35 day. Symbols represent the different maturity stages are (Zibdia: -{- M1, -- M2 and -U-
M3) and (Hindi: - - M1, - - M2, and -▲- M3). Solid lines represent linear regression.
Another index of cell injury is ion leakage, which is a commonly used technique to
assess cell damage or viability. The detail of the effect of storage temperature on
the increase of ion leakage with time has been described previously (Chapter 3).
Ion leakage correlates well with the accumulation of MDA equivalents (Chapter 4),
and as MDA equivalents are formed from membrane oxidation this link may be
direct. In spite of the expected similarity in the correlations obtained between
145
Chapter 5
enzyme activity and MDA equivalents, it was thought valuable to consider how
changes in anti-oxidative enzyme activity might correlate with ion leakage.
PEEL
1.8
ZIBDIA HINDI
y = -0.0103x + 0.9579 y = -0.0097x + 1.0145
1.6 4oC R2 = 0.6241 4oC R2 = 0.4096
y = -0.0139x + 1.3294
1.4 y = -0.0139x + 1.3977
R2 = 0.5311
R2 = 0.3295
1.2 y = -0.0157x + 1.5948
y = -0.0138x + 1.5556
1.0 R2 = 0.5057
R2 = 0.2597
0.8
0.6
0.4
0.2
0.0
1.8
y = -0.0194x + 1.2718
10oC R2 = 0.8159 10oC
y = -0.0195x + 1.3578
1.6 R2 = 0.5203
1.4
1.2 y = -0.0214x + 1.8211
R2 = 0.7231 y = -0.0129x + 1.425
1.0 R2 = 0.2788
0.8
0.6
0.4
y = -0.0181x + 1.4345 y = -0.0117x + 1.5086
0.2 R2 = 0.1433
R2 = 0.6709
0.0
0 20 40 60 80 0 20 40 60 80
APX activity in peel (µM g-1 FW)
Figure 10a. The relationship of malondialdehyde (MDA) content in function of ascorbate peroxidase
(APX) activity on fruits peel of Zibdia and Hindi varieties harvested in different maturity stages were
regression.
The inverse correlation between ion leakage and CAT activity is good, especially
for Hindi peel and pulp tissues (Figure 15). Likewise the correlation between APX
activity and ion leakage was also good (Figure 14); comparable to that shown
between APX activity and the accumulation of MDA equivalents, with decreased
activities of APX correlating with increased leakage (Figure 16 and 17). The activity
146
Antioxidant Enzymes
of neither GR nor SOD correlated with the increase of ion leakage that occurred
during storage.
PULP
ZIBDIA HINDI
1.8
o
1.6 4 C y = -0.0136x + 1.1004 4oC
R2 = 0.6544
1.4
1.2
1.0
0.8
MDA content of fruit pulp (ηM g-1 FW)
0.6 y = -0.0128x + 1.0363

R2 = 0.3333
0.4
y = -0.0171x + 1.4868 y = -0.0165x + 1.5292
y = -0.0069x + 1.4728 y = -0.017x + 1.7561
0.2 R2 = 0.6089
R2 = 0.0881 R2 = 0.5503 R2 = 0.6113
0.0
1.8
1.6
y = -0.0066x + 0.8111
2
10oC 10oC
R = 0.2509 y = -0.0135x + 1.2393
1.4 R2 = 0.4973
1.2
1.0
0.8
0.6
y = -0.0106x + 1.1394
0.4 2
R = 0.4775 y = -0.0095x + 1.3801
y = -0.0152x + 1.5906 y = -0.0136x + 1.8008
0.2 2 R2 = 0.1645
R = 0.4152 R2 = 0.2948
0.0
0 20 40 60 80 0 20 40 60 80
Figure10b. The relationship of malondialdehyde (MDA) content in function of ascorbate peroxidase

(APX) activity on fruits pulp of Zibdia and Hindi varieties harvested in different maturity stages were
regression.
147
Chapter 5
PEEL
ZIBDIA HINDI
1.8 y = -0.0213x + 0.8782
o o y = -0.0297x + 1.0502
1.6 4 C R2 = 0.4571 4 C R2 = 0.7062
1.4
1.2 y = -0.0309x + 1.2103 y = -0.0393x + 1.3879
R2 = 0.5086 R2 = 0.7488
1.0
0.8
0.6
0.4
0.2 y = -0.0377x + 1.5891 y = -0.0209x + 1.2347
R2 = 0.6263 R2 = 0.4254
0.0
1.8 y = -0.031x + 0.929
o o y = -0.0371x + 1.2913
1.6 10 C R2 = 0.6415 10 C R2 = 0.7836
1.4
y = -0.0355x + 1.1304
1.2
R2 = 0.7224
y = -0.0473x + 1.6126
1.0
R2 = 0.8938
0.8 y = -0.0301x + 1.1956
R2 = 0.5969
0.6
0.4
y = -0.0469x + 1.7831
0.2
R2 = 0.7814
0.0
0 10 20 30 40 0 10 20 30 40
CAT activity in peel (µM g-1 FW)
Figure 11a. Presents the relationship of malondialdehyde (MDA) content in function of Catalase
(CAT) activity on fruits peel of Zibdia and Hindi varieties harvested in different maturity stages were
regression.
148
Antioxidant Enzymes
PULP
ZIBDIA HINDI
1.8
o y = -0.03x + 0.9483
o y = -0.0528x + 1.2748 4 C
1.6 4 C R2 = 0.8223 R2 = 0.7224
1.4
1.2 y = -0.0609x + 1.7907 y = -0.0353x + 1.2173

1.0 R2 = 0.4678 R2 = 0.809
0.8
0.6
0.4
y = -0.0225x + 1.2443
0.2 y = -0.0485x + 2.099 R2 = 0.6893
R2 = 0.4764
0.0
1.8
y = -0.0277x + 0.8313 o y = -0.0374x + 1.0555
1.6 10oC R2 = 0.6448
10 C R2 = 0.6519
1.4
1.2 y = -0.0295x + 1.4171
y = -0.027x + 0.9558 R2 = 0.4318
1.0
R2 = 0.6506
0.8
0.6
0.4
y = -0.0272x + 1.2248 y = -0.0322x + 1.7417
0.2
R2 = 0.6808 R2 = 0.4789
0.0
0 10 20 30 40 0 10 20 30 40
Figure 11b. The relationship of malondialdehyde (MDA) content in function of Catalase (CAT)
activity on fruits pulp of Zibdia and Hindi varieties harvested in different maturity stages were stored
at 4 and 10oC for 35 day. Symbols represent the different maturity stages are (Zibdia: -{- M1, --
M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3). Solid lines represent linear
regression.
149
Chapter 5
PEEL
ZIBDIA HINDI
1.8
o
1.6
o
4 C y = 0.0086x + 0.3847 4 C y = -0.0033x + 0.6828
R2 = 0.336 R2 = 0.0292
1.4
1.2 y = 0.0047x + 0.5508

R2 = 0.0867 y = -0.0006x + 0.7286
1.0 R2 = 0.0017
0.8
0.6
0.4
y = 0.0033x + 0.6371 y = -0.0004x + 0.8008
0.2 R2 = 0.0533 R2 = 0.0009
0.0
1.8
o o
1.6 10 C y = -0.0041x + 0.6601 10 C y = -0.0131x + 0.9172
R2 = 0.0535 R2 = 0.1369
1.4
1.2
1.0 y = -0.01x + 0.9787

y = -0.003x + 0.7176
R2 = 0.0189 R2 = 0.0924
0.8
0.6
0.4
y = -0.0087x + 1.0426
0.2 y = -0.0046x + 0.8433 R2 = 0.0713
R2 = 0.0337
0.0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 12a. The relationship of malondialdehyde (MDA) content in function of glutathione reductase
(GR) activity on fruits peel of Zibdia and Hindi varieties harvested in different maturity stages were
regression.
150
Antioxidant Enzymes
PULP
ZIBDIA HINDI
1.8
o
4 C y = 0.0113x + 0.4408 o y = 0.0041x + 0.5822
1.6 R2 = 0.1824
4 C R2 = 0.0302
1.4
1.2
y = -0.0016x + 0.8114
1.0 R2 = 0.0071
0.8
0.6 y = 0.0126x + 0.5265

0.4 R2 = 0.1257
y = -0.003x + 0.9806
0.2 y = 0.0145x + 0.7226
R2 = 0.0735
R2 = 0.2148
0.0
1.8
o o y = -0.0037x + 0.7503
1.6 10 C y = 0.015x + 0.385 10 C R2 = 0.0263
R2 = 0.1803
1.4
y = -0.0003x + 0.9194
1.2
R2 = 0.0005
y = 0.0109x + 0.4736
1.0 R2 = 0.1587
0.8
0.6
0.4
y = 0.0072x + 0.6519 y = -0.0022x + 1.1351
0.2 R2 = 0.0474 R2 = 0.0305
0.0
0 10 20 30 40 50 0 20 40 60 80
Figure 12b. The relationship of malondialdehyde (MDA) content in function of glutathione reductase
(GR) activity on fruits pulp of Zibdia and Hindi varieties harvested in different maturity stages were
regression.
151
Chapter 5
PEEL
ZIBDIA HINDI
1.8
o
1.6
y = -0.0044x + 0.6865 4oC y = -0.0161x + 1.0616 4 C
R2 = 0.0438 R2 = 0.3406
1.4
1.2
y = -0.0166x + 1.1776
1.0 R2 = 0.1129
0.8
y = -0.0254x + 1.4463
0.6 R2 = 0.2182
0.4
0.2 y = -0.0337x + 1.8811 y = -0.0367x + 1.9127
R2 = 0.1014 R2 = 0.2388
0.0
1.8
o o
1.6
y = -0.0041x + 0.6915 10 C y = -0.0268x + 1.4339 10 C
R2 = 0.0069 R2 = 0.1632
1.4
1.2
y = -0.0168x + 1.2967
1.0 y = -0.0158x + 1.0949 R2 = 0.0823
R2 = 0.0449
0.8
0.6
0.4
y = -0.0033x + 0.99
0.2 y = -0.0698x + 2.9217 R2 = 0.0013
R2 = 0.3814
0.0
0 10 20 30 40 0 10 20 30 40
Figure 13a. The relationship of malondialdehyde (MDA) content in function of superoxide reductase
(SOD) activity on fruits peel of Zibdia and Hindi varieties harvested in different maturity stages were
regression.
152
Antioxidant Enzymes
PULP
ZIBDIA HINDI
1.8
1.6 y = -0.003x + 0.6977 4oC o

4 C
R2 = 0.0123 y = -0.011x + 0.9223
1.4 R2 = 0.2573
y = -0.0139x + 1.1847
1.2
R2 = 0.071
1.0
0.8
y = -0.0167x + 1.2644
0.6
y = 0.0016x + 1.0755 R2 = 0.1779
0.4 R2 = 0.0006
y = -0.0221x + 1.5621
0.2
R2 = 0.131
0.0
1.8
o
1.6 y = -0.005x + 0.6705 10 C y = -0.0277x + 1.3249
10oC
R2 = 0.0433 R2 = 0.4114
1.4
1.2
y = -0.0151x + 1.0697
1.0 R2 = 0.1714
0.8
y = -0.0145x + 1.2869
0.6
y = -0.0206x + 1.4452 R2 = 0.0692
0.4 R2 = 0.1518
y = 0.0003x + 1.0393
0.2
R2 = 4E-05
0.0
0 10 20 30 400 10 20 30 40
SOD activity of frits pulp (unit g-1 FW)
Figure 13b. The relationship of malondialdehyde (MDA) content in function of superoxide reductase
(SOD) activity on fruits pulp of Zibdia and Hindi varieties harvested in different maturity stages were
regression.
153
Chapter 5
PEEL
80
ZIBDIA HINDI
y = -0.8273x + 66.918 y = -0.3043x + 51.299
o o
4 C R2 = 0.4961 4 C R2 = 0.483
60 y = -0.3005x + 60.464
R2 = 0.5016
40
y = -0.6953x + 71.34
20 R2 = 0.6989
y = -0.2297x + 65.937 y = -0.2717x + 66.883

R2 = 0.2321 R2 = 0.3698
0
80
y = -0.5101x + 56.99 o y = -0.3019x + 44.956
10oC R2 = 0.7226
10 C
R2 = 0.5892
60
y = -0.326x + 62.43
y = -0.6378x + 70.562 R2 = 0.4083
R2 = 0.6404
40
20
y = -0.6836x + 79.814 y = -0.286x + 64.914

R2 = 0.742 R2 = 0.1623
0
0 20 40 60 80 100 0 20 40 60 80 100
Ion leakage % of fruits peel
Figure 14a. The linear relationship of ascorbate peroxidase (APX) activity in function of ion leakage
percentage of fruits peel of two mango cultivars ‘Zibdia‘ and ‘Hindi’ harvested in different maturity
stages: immature (M1), half-mature (M2) and full mature fruits (M3), stored at 4 and 10oC for 35 day.
Enzymes activity measured on peels, versus storage time at 5 different storage temperatures.
Symbols represent the different maturity stages are (Zibdia: -{- M1, -- M2 and -U- M3) and (Hindi:
- λ - M1, - ν - M2, and -▲- M3). The lines represent linear regression.
154
Antioxidant Enzymes
PULP
80
ZIBDIA HINDI
o y = -0.4831x + 55.28 o
4 C 2
R = 0.4563
4 C y = -0.4627x + 60.933
R2 = 0.6306
60
40
y = -0.6506x + 75.115
R2 = 0.6835
20 y = -0.5297x + 68.899
R2 = 0.5296
y = -0.4422x + 78.022 y = -0.4536x + 74.075
2
R = 0.4481 R2 = 0.5552
0
80
o y = -0.4547x + 66.53
10 C y = -0.5442x + 69.314
10 C
o 2
R2 = 0.3062 R = 0.5596
60
40
y = -0.2739x + 66.072
y = -0.5407x + 77.519 2
R = 0.2882
20 R2 = 0.5437
y = -0.4027x + 77.124 y = -0.3227x + 73.668

R2 = 0.3746 2
R = 0.4226
0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 14b. The linear relationship ascorbate peroxidase (APX activity in function of ion leakage
Symbols represent the different maturity stages are (Zibdia: -{- M1, -- M2 and -U- M3) and
(Hindi: - λ - M1, - ν - M2, and -▲- M3). The lines represent linear regression.
155
Chapter 5
PEEL
ZIBDIA HINDI
40
o y = -0.3652x + 28.137 o
4 C 2
R = 0.7289 4 C y = -0.1631x + 20.731
R2 = 0.7477
y = -0.2918x + 27.499
30 R2 = 0.6311
y = -0.2023x + 24.676
20 R2 = 0.8047
10
y = -0.1483x + 30.419 y = -0.2584x + 32.026

R2 = 0.4507 R2 = 0.4705
0
40
o o
10 C y = -0.2139x + 20.159 10 C y = -0.2052x + 24.05
R2 = 0.411 R2 = 0.6513
30 y = -0.2271x + 27.618
y = -0.3374x + 27.934 2
R = 0.8266
R2 = 0.64
20 y = -0.3379x + 33.001
2
y = -0.4115x + 32.841 R = 0.6627
R2 = 0.6457
10
0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 15a. The relationship of Catalase (CAT) activity in function of ion leakage percentage of fruit
peel of two mango cultivars ‘Zibdia‘ and ‘Hindi’ harvested in different maturity stages: immature
(M1), half-mature (M2) and full mature fruits (M3), stored at 4 and 10oC for 35 day. Symbols
represent the different maturity stages are (Zibdia: -{- M1, -- M2 and -U- M3) and (Hindi: - λ -
M1, - ν - M2, and -▲- M3). The lines represent linear regression.
156
Antioxidant Enzymes
PULP
ZIBDIA HINDI
40
o y = -0.2509x + 24.326
4 C 2
o
4 C
y = -0.2318x + 23.777
R = 0.8491 R2 = 0.6995
30
y = -0.1268x + 23.194
2
R = 0.5038
20 y = -0.3833x + 29.969
R2 = 0.735
10
y = -0.1884x + 31.773 y = -0.3419x + 33.09

2
R = 0.7498 R2 = 0.4916
0
40
o o
10 C y = -0.3095x + 26.623 10 C y = -0.2145x + 30.265
2
R = 0.6741 R2 = 0.6461
30
y = -0.2487x + 25.776 y = -0.1889x + 20.517
2
R = 0.5443 R2 = 0.5668
20
10
y = -0.4159x + 41.301 y = -0.1965x + 32.686
2
R = 0.7772 R2 = 0.5407
0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 15b. The relationship of Catalase (CAT) activity in function of ion leakage percentage of
fruits pulp of two mango cultivars ‘Zibdia‘ and ‘Hindi’ harvested in different maturity stages:
immature (M1), half-mature (M2) and full mature fruits (M3), stored at 4 and 10oC for 35 day.
157
Chapter 5
PEEL
ZIBDIA HINDI
70
4oC y = 0.2456x + 13.268 o
4 C
y = -0.1069x + 17.211
R2 = 0.0719 R2 = 0.0933
60
y = -0.1592x + 28.679
50 R2 = 0.0493
40 y = -0.2135x + 43.887
R2 = 0.0437
30
20 y = 0.1493x + 27.257
R2 = 0.0227
10 y = 0.3567x + 20.632
R2 = 0.2303
0
70
o y = -0.1483x + 19.352
10 C y = -0.2635x + 29.224
2
10oC R2 = 0.2413
60 R = 0.13
y = -0.0714x + 26.392 y = -0.1639x + 23.763

50 2 R2 = 0.1875
R = 0.0076
40 y = -0.2347x + 27.871
y = -0.1358x + 32.094
2
R2 = 0.1204
R = 0.0285
30
20
10
0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 16a. The relationship Glutathione reductase (GR) activity in function of ion leakage
158
Antioxidant Enzymes
PULP
ZIBDA HINDI
50 y = 0.0323x + 9.6725
o y = 0.1326x + 9.8684 o
4C R2 = 0.0493 4 C R2 = 0.0061
40 y = -0.2191x + 42.54
y = 0.116x + 13.319 R2 = 0.0333
R2 = 0.0669
30
20
y = -0.2547x + 30.342
R2 = 0.075
10
y = 0.0137x + 26.912
R2 = 0.0008
0
50
o y = 0.1259x + 3.7221 o y = -0.1393x + 22.561
10 C 10 C 2
R = 0.0747
R2 = 0.1177
40
y = 0.1297x + 8.062
30
R2 = 0.0986
20
y = -0.1473x + 37.341
2
10 R = 0.0321
y = -0.2474x + 54.954
y = 0.118x + 11.759 2
R2 = 0.063 R = 0.0602
0
0 20 40 60 80 100 0 20 40 60 80 100
Ion leakage % of fruits pulp
Figure 16b. The relationship of Glutathione reductase (GR) activity in function of ion leakage
percentage of fruits pulp of two mango cultivars ‘Zibdia‘ and ‘Hindi’ harvested in different maturity
159
Chapter 5
PEEL
ZIBDIA HINDI
50
4oC y = -0.3225x + 37.05 o
4 C
y = -0.1891x + 34.185
R2 = 0.2472 R2 = 0.6126
40
y = -0.1014x + 34.824
R2 = 0.3969
30
20
y = -0.1604x + 34.484
10
R2 = 0.2488
y = -0.0219x + 34.148 y = -0.1246x + 33.406
R2 = 0.0486 R2 = 0.438
0
50
o o y = -0.0891x + 32.709
10 C y = -0.0372x + 27.251 10 C
R2 = 0.0201 R2 = 0.1744
40
y = -0.1104x + 36.118
R2 = 0.3899
30
20
y = -0.076x + 28.891
R2 = 0.2238
10 y = -0.0393x + 29.863 y = -0.0217x + 33.374

R2 = 0.0279 R2 = 0.0083
0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 17a. The relationship of Superoxide dismutase (SOD) activity in function of ion leakage
160
Antioxidant Enzymes
PULP
ZIBDIA HINDI
50
o y = -0.2265x + 39.413
4 C y = -0.1863x + 33.458
o
4 C R2 = 0.2531
2
40 R = 0.0993
y = -0.2155x + 38.822
R2 = 0.2364
30
20
SOD activity of fruit pulp (unit g-1 FW)
y = -0.1281x + 36.508
2
R = 0.1761
10
y = -0.0893x + 37.587 y = -0.0902x + 35.139
2
R = 0.1458 R2 = 0.1736
0
50
o o y = -0.0749x + 30.497
10 C y = -0.2473x + 38.961 10 C
R2 = 0.2107 R2 = 0.1194
40
30
20
y = -0.1522x + 36.954
R2 = 0.2425
10 y = -0.1884x + 33.545
y = -0.11x + 38.524
R2 = 0.491 y = -0.0132x + 27.831
R2 = 0.1403
R2 = 0.0024
0
0 20 40 60 80 100 0 20 40 60 80 100
Figure 17b. The relationship of Superoxide dismutase (SOD) activity in function of ion leakage
percentage of fruits pulp of two mango cultivars ‘Zibdia‘ and ‘Hindi’ harvested in different maturity
Acknowledgment
The author was supported by a grant from the Egyptian High Educational Ministry.
We thank Dr Jan W. Rijstenbil of Department Marine Microbiology Netherlands
Institute of Ecology Centre for Estuarine and Marine Ecology for helping in
measurement of antioxidant enzymes.
161
Chapter 5
162
Chapter 6
On the Ethylene production, respiration rate,
firmness and ion leakage in mango fruits

*
Horticultural Production Chains Group, Marijkeweg 22, 6709 PG Wageningen
University. The Netherlands
Chapter 6
Abstract
Physiological changes such as: ethylene production, respiration rate, firmness, and
ion leakage were studied on two mango varieties (Mangifera indica L. ‘Zibdia’ and
‘Hindi Be-Sennara’). These changes were monitored at 3 different harvest maturity
stages for fruits stored for up to 35 days at different temperatures (2, 4, 7, 10, and
20oC). C2H4 production increased to a maximum production rate after the 5th day of
storage. The climacteric respiration showed a peak after day 10. Fully mature (M3)
fruits produced more ethylene and respiration compared to immature (M1) and half
mature fruits (M2). Thereafter they decreased until day 35. Hindi fruits show higher
firmness than Zibdia fruits. Both mango varieties show higher firmness at low
storage temperature (2 or 4oC) compared with fruits stored at 7 or 10oC. Ion
leakage in Zibdia fruits was higher than Hindi fruits when the fruits were stored at 2
or 4oC compared with fruits stored at 7 or 10oC. Fruit pulps showed higher ion
leakage than peel. Decreased firmness and increased ion leakage due to ripening
processes when fruits were stored at 20oC which are related to changes in cell lipid
membrane. Physiological changes are induced by the severity effects of low
storage temperatures at different storage times. At lower temperature, storage
induced CI in both varieties as an effect of generation active oxygen species
(AOS). These AOS react with cell lipid membranes and initiate lipid peroxidation.
Because of these processes, chilling injury will result which affects the quality
elements of mango fruits.
Keywords: chilling injury (CI); Ethylene (C2H4); Respiration (CO2); Firmness (Fr); Electrolyte leakage
(EL)
164
Ethylene, Respiration, Firmness and Ion Leakage
1. Introduction
Mango is one of the most popular fruits worldwide (Mitra and Baldwin 1997)
and also very popular in Egypt (Mamdouh, 1997). It is considered to be a
climacteric fruit which ripens rapidly after harvest, and to be a moderate producer of
C2H4 and CO2 (Kader, 2002). Disease susceptibility, sensitivity to low storage
temperature below 12oC, and perishability due to ripening and softening limit the
storage, handling and transport potential of the fruit (Mitra and Baldwin, 1997).
Avoidance of prolonged exposure to low temperatures is considered an important
requirement for this fruit as it is chilling sensitive. As a result of this sensitivity, low
temperatures produce a range of physiological and metabolic disorders that lead to
serious quality losses. The various dysfunctions that arise under low temperature
conditions result in various physical and metabolic changes that are easily scored
and which can therefore be used to assess the degree of chilling injury.
Mango is a climacteric fruit. Measurements on the mango variety ‘Manila’
showed that a typical climacteric respiration peak develop by the 6th day of storage
and decreased steadily thereafter (Dinora et al., 1997). Other climacteric
respiratory patterns have been observed in the varieties ‘Tommy Atkins’ by
Mitcham and McDonald, (1992), and ‘Kent’ by Trinidad et al. (1997). The maximum
of C2H4 evolution occurred simultaneously with the increase of respiration in the
variety ‘Haden’ (Trinidad et al., 1997). On the other hand, McCollum (1993) found
that for the variety ‘Kent’ C2H4 production reached a maximum after the climacteric
of respiration. These results indicated that the degree of synchronization of C2H4
evolution with the respiratory climacteric depends upon the mango variety.
Various factors influence the production of ethylene. Low temperatures during
long term storage inhibit C2H4 production in fruits, including mangoes (Valero et al.,
1997). Besides this, the maturity of mango fruits affects the rate of C2H4 production:
fully mature fruits produce larger amounts of C2H4 than immature and half mature
fruits (Majeed and Jeffery, 2002). Physiologically, the effects of ethylene on the
ripening and senescence of plants is correlated well with climacteric respiration and
changes in many membrane parameters, such as loss of phospholipids and
increased membrane permeability (Noodén and Leopold, 1988). The decrease of
climacteric respiration rates at low storage temperatures is a result of decreased
metabolic activity. This is considered a secondary response to low temperatures.
These changes in C2H4 production and respiration rates are correlated with major
changes in the properties of the fruits. For example, various hydrolytic enzymes
that degrade cell wall components are released and their activity is correlated with
changes in fruit tissue firmness (Ketsa et al., 1999b). Overall, changes in fruit and
vegetable firmness are a consequence of physical, physiological, and chemical
changes that occur in the tissues of the product. The processes associated with
165
Chapter 6
changes in firmness have been intensively studied because firmness is considered

to be an important factor determining the consumer acceptability of fruits. Fruit
firmness is a set of physical characteristics that are sensed by means of touch, and
are related to the deformability or disintegration of the fruit tissues, and the flow of
tissue fluids in response to the application of a force. It is possible to measure the
response of a fruit to an applied force in terms of the time dependent development
of a deformation and use this to describe objectively the phenomena that result in
the sensory evaluation of firmness (Bourne, 1980).
The processes that changes firmness, and thus product itself firmness, are
influenced by the product’s history and immediate environment. For example,
storage temperature is one of the factors that affects changes in the firmness of
‘Keitt’ mango fruits. Lederman et al., (1997) found that storage of these fruits at low
temperature improved the firmness. Owing to their chilling sensitivity, low storage
temperature produces an increased membrane permeability in mango. Firmness,
however, depends not only on cell-wall properties, which are often studied, but also
on membrane integrity. Without membrane integrity it is impossible to generate the
turgor forces that give rise to many of the mechanical properties of thin-celled
tissues, such as fruit pulp. So, an aim of this study was to see if damage to the cell
membranes of the pulp tissue resulting from storage at low temperatures was
correlated with changes in tissue mechanical properties.
Tissue softening is one of the consequences of ethylene exposure. So, in
addition to tissue firmness, we also measured changes in ethylene evolution and
respiration to investigate the effect of storage at chilling temperatures on the
climacteric of mangoes. These measurements were made on two varieties of
mango: Zibdia and Hindi, which were harvested at three different maturity stages
and stored at different temperatures for up to 35 days.
2.1. Fruits and storage condition:

Zibdia and Hindi Be-Sennara mango fruits were harvested from trees more
than 20 years old. (Details of harvesting, transporting and shipping are given
previously in Chapter 3). Upon arrival in PPO, the fruits were divided into 5
batches; each one composed of 216 Zibdia and 216 Hindi (72 fruits of each
maturity stage). These batches were stored at 2, 4, 7, 10 and 20oC in darkness.
Samples of 10 fruits were taken from each temperature every 5 days (up to 35
days) and used for measurements of CO2 and ethylene evolution, electrolyte
leakage, the accumulation of malondialdehyde equivalents, and tissue firmness.
2.2. Ethylene production and respiration rate:
166
Ethylene and respiration rates of Zibdia and Hindi fruits were measured on
samples of 9 fruits after 5 days interval for each maturity stage. Each sample was
divided into 3 replicates of 3 fruits. Individual fruits were inserted in 1-liter glass jars,
and incubated for 1h at each storage temperature. A sample of headspace
atmosphere was collected and analyzed for ethylene and carbon dioxide by gas
chromatography techniques (GC). These fruits were then used for destructive
measurements (firmness and ion leakage). Ethylene was measured using a GC,
GC 6000 Vega Series, Carlo Erba Instrument, (Milan, Italy) and carbon dioxide was
also measured by GC techniques (Micro GC, Chrompack, Middelburg, The
Netherlands)
2.3. Firmness measurement:

Fruit firmness was determined on the same fruits which were used previously
in ethylene and respiration measurements. A Zwick® Universal Testing Machine
with a 6.35-mm-diameter 60o conical probe was used to perform these
measurements. The instrument measures the force required for the mechanical
probe to penetrate 5.5 mm into the tissue of fruits at a speed of 3 mm s-1. The
measurements were carried out according to the standard procedure, in the centre
of the on two opposite faces of the fruit. The measured forces (in Newtons) were
then averaged (Hofman et al., 1997).
2.4. Electrolyte Leakage
Changes in electrolyte leakage was described and measured in Chapter 3
Changes lipid peroxidation of cell membrane product (malondialdehyde;

MDA) concentration was described and measured in Chapter 4

Measured data for ethylene, respiration, production, firmness and ion leakage
were analyzed using analysis of variance (ANOVA). Four factors were included:
time, temperature, variety and maturity stages. Means comparisons were
undertaken using the least significant differences (L.S.D.) at p=0.05. Data were
analyzed for two periods: the first is from 0-20 days, when all treatments were
present and the second is from day 0-35 days for all treatments except for storage
at 20oC. Linear regression analysis and ANOVA were analyzed at the 5%
167
Chapter 6
probability level. The statistical software package Genstat 8 (Lawes Agricultural

Trust, Rothamsted Experimental Station, UK) was used.
3. Results
3.1. Ethylene production
Figure 1 shows the changes in ethylene production rate as a function of
storage time (days) for both mango varieties at different storage temperatures (2-
20oC) for three maturity stages. Generally, the rate of ethylene production depends
in a complex way on the storage time, temperature, variety and maturity stage.
Both mango types show almost the same initial rate of ethylene production:
approximately 10ml.kg-1.h-1 at all the maturity stages. During storage in the
temperature range 2-20oC, ethylene evolution increases to a maximum that is
higher than the initial rate of evolution by 2-7 times for Zibdia and 2-6 times for
Hindi. The maximum is reached after 5 days of storage at all the temperatures and
for all the maturity stages. For Zibdia the maximum ethylene evolution occurred in
the 20°C fruits, and was substantially higher than the evolution from the fruits held
at low storage temperatures. These latter fruits displayed a temperature sensitivity
of the rate of maximum evolution with evolution increasing with increasing
temperature. The effect of maturity stage on the climacteric of Hindi fruits was less
marked than for Zibdia. In contrast to Zibdia, storage at 10°C did not decrease
ethylene evolution during the climacteric compared to the fruits stored at 20°C, and
with increasing maturity stage the rate of evolution from the 7°C fruits increased to
that of the 10° and 20°C fruits. Evolution from the 2° and 4°C fruits was similar to
each other and increased with maturity stage.
The maximum of ethylene evolution was followed by a period of decreasing
ethylene production that differs depending mango type, and storage temperature.
The ethylene evolution of Zibdia fruits stored at low temperatures decreased in
parallel, with little difference between the temperatures, to a very low level at day
35. These differences parallel the variation in the maximum ethylene evolution of
the fruits. The decrease of evolution from the maximum in Hindi fruits also led to a
very low rate of evolution at day 35, and as with Zibdia M3 fruits, the decay curves
show temperature sensitivity that parallels the temperature sensitivity of the
maximum rate of evolution.
168
90 ZIBDIA HINDI
M1 M1
60
30
0
90
M2 M2
60
C2H4 concentration, mg Kg-1 h-1
30
0
90
M3 M3
60
30
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure1. Ethylene production versus storage time of two mango varieties Zibdia and Hindi
harvested at different maturity stages: immature (M1), half-mature (M2), and full mature fruits (M3)
as a function of storage time at different storage temperatures. Symbols represent the storage
7, -¡- 10 and -«-200C). CO2 expressed as mean (n=3).The Vertical bars indicate L.S.D. at p=0.05
in two periods: the first period (0-20 days; all treatments present) and the second (0-35 days; when
all treatments were present except for storage at 20oC).
169
Chapter 6
3.2. Respiration rate

Figure 2 shows the climacteric respiration rates of both mango varieties
measured by CO2 production in mg.Kg-1.h-1 plotted as a function of storage time at
different storage temperatures for both mango varieties at three different maturity
stages. The interaction at p<0.001 was significant interaction among storage time
(days), temperatures, maturity and varieties. On day zero, the respiration rate was
found to be slightly higher in Zibdia than Hindi fruits at all maturity stages (~4 vs ~8
mg kg-1 h-1). Both varieties show a slight decrease in the respiration rate up to the
fifth storage day independent of the storage temperature and/or the maturity stage.
It can be seen from Figure 2 that the respiration rate shows a sudden large
increase from day 5 until day 10 of storage time for both mango varieties at all
maturity stages. This is followed by a sudden decrease with almost the same rate
to reach similar values to that found at day 5 in all cases. This peak is very large
when the fruits are stored at 20oC. Starting from day 15, the respiration rate
increases slightly up to 35 days in M2 and M3 in both fruit types. For M3 fruits, the
respiration rate decreases again after 30 days of storage. The variation of the
respiration rate is found to be temperature dependent.
3.3. Firmness
Figure 3 shows the fruit firmness (N) of both mango varieties plotted as a
function of storage time (days) at different storage temperatures for both mango
varieties at three different maturity stages. The interaction at p<0.001 was
significant among storage time (days), temperatures, maturity and varieties. The
results show that Hindi fruits are initially firmer than Zibdia. Considering Hindi fruits
in the different maturity stages, it is clear that the immature fruits are always more
firm (160 N) than the half mature (140 N) and fully mature fruits (130 N). On the
other hand, the firmness in Zibdia is approximately the same (~120 N) at all three
maturity stages. Fruit firmness decreases progressively during the storage period
up to the end of the experiment. Two different rates of decrease in fruit firmness
can be seen at 20oC in Zibdia fruits. In M1 Zibdia fruits, a fast decrease was seen
up to day 10 of storage followed by a slight decrease up to day 20, where it
reached its lowest level. M2 fruits showed a modest decrease in the fruit firmness
in the first 15 days followed by larg drop to a low level up to the day 20. M3
firmness decreased with a high constant rate to its lowest level at day 20. For Hindi
fruits stored at the same temperature (20oC), M1 and M2 showed decreasing fruit
firmness at a more or less constant rate up to day 20. M3 Hindi fruits reached their
lowest firmness value after 10 days of storage. No significant differences can be
seen between and in them M1 and M2 Zibdia fruits stored at 2-10oC during the first
170
ZIBDIA HINDI
160
M1 M1
120
80
40
0
160
M2 M2
CO2 evolution mg Kg-1 h-1
120
80
40
0
160
M3 M3
120
80
40
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 2. Carbon dioxide rate versus storage time of two mango varieties Zibdia and Hindi
harvested at different maturity stages: immature (M1), half-mature (M2), and full mature fruits (M3)
as a function of storage time at different storage temperatures. Symbols represent the storage
7, -¡- 10 and -«-200C). CO2 expressed as mean (n=3).The Vertical bars indicate L.S.D. at p=0.05
in two periods: the first period (0-20 days; all treatments present) and the second (0-35 days; when
all treatments were present except for storage at 20oC).
171
Chapter 6
160 ZIBDIA HINDI

M1 M1
120
80
40
0
160
M2 M2
120
Firmness (N)
80
40
0
160
M3 M3
120
80
40
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 3. Firmness versus storage time of two mango varieties Zibdia and Hindi harvested at
different maturity stages: immature (M1), half-mature (M2), and full mature fruits (M3) as a function
of storage time at different storage temperatures. Symbols represent the storage temperatures are
(Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: - z - 2, - - 4, -▲- 7, -¡- 10 and -«-
200C). Firmness expressed as mean (n=3).The Vertical bars indicate L.S.D. at p=0.05 in two
periods: the first period (0-20 days; all treatments present) and the second (0-35 days; when all
treatments were present except for storage at 20oC).
172
10 days of storage time. The firmness of Zibdia fruits is inversely proportional to the
storage temperature. The effect is more pronounced in M1 and M2 fruits in
comparison to M3 fruits, and especially in fruits stored at 10oC. The same behavior
was also found for Hindi fruits where, at 10oC, a more significant decrease started
from day 15 in M1 and M2 and day 10 in M3.
2.4. Electrolyte Leakage
Changes in electrolyte leakage was described in Chapter 3 (Figure 4)
3.5. Lipid peroxidation measured as the formation of MDA equivalents
Changes in lipid peroxidation of cell membranes, caused by low storage

temperature, give rise to products such as malondialdehyde (MDA) in mango fruits
as described in chapter 3 (Figure 5).
4. Discussion
4.1. Ethylene production (C2H4)
Ethylene is a simple molecule which has profound effects on many aspects

of the growth and development of plants. Plant tissues of all ages can be induced
to produce ethylene in response to various challenges, and plant tissues will
respond to ethylene in a range of ways. The evolution of ethylene and the response
of tissues to applied ethylene are, however, under developmental control: they are
to various degrees dependent upon tissue type and age. So, ethylene synthesis
occurs at specific times in the developmental trajectory of certain tissues, for
example during fruit ripening, leaf and flower senescence and abscission. A wide
range of stresses will also provoke ethylene production, for example mechanical
trauma such as bruising and cutting, temperature stresses, and chemical stress
(Heun-Hong and Kenneth, 2000).
During storage, mango fruits show an initial maxima in ethylene production
after day 5 of storage at all maturity stages of both varieties. This result is
consistent with previous studies. The increase in C2H4 production rate at the day 5
may be due to an increased reaction rate of s-adenosyl-methionine (SAM) to 1-
cyclopropane-1-carboxylic acid (ACC), which is an immediate precursor of
ethylene, a reaction catalyzed by ACC synthase and ethylene forming enzymes, as
recently reviewed by Baldwin (2004). Thereafter, the ethylene production rates
decline gradually up to day 15 of storage in all maturity stages of both mango
varieties. Storage temperature appears to be another determining factor for the
formation of C2H4, and possibly the activity of ACC synthase.
173
Chapter 6
PEEL
100
ZIBDIA HINDI
M1 M1
80
60
40
20
0
100
M2 M2
80
60
40
20
0
100
M3 M3
80
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 4a. Ion leakage percentage of fruits peel versus storage time of two mango varieties Zibdia
- 4, -▲- 7, -¡- 10 and -«-200C). Ion leakage expressed as mean (n=3).The Vertical bars indicate
L.S.D. at p=0.05 in two periods: the first period (0-20 days; all treatments present) and the second
(0-35 days; when all treatments were present except for storage at 20oC).
174
PULP
ZIBDIA HINDI
120
M1 M1
100
80
60
40
20
0
120
M2 M2
100
80
60
40
20
0
120
M3 M3
100
80
60
40
20
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 4b. Ion leakage percentage of fruits pulp versus storage time of two mango varieties Zibdia
- 4, -▲- 7, -¡- 10 and -«-200C). Ion leakage expressed as mean (n=3).The Vertical bars indicate
L.S.D. at p=0.05 in two periods: the first period (0-20 days; all treatments present) and the second
(0-35 days; when all treatments were present except for storage at 20oC).
175
Chapter 6
PEEL
ZIBDIA HINDI
2.0
M1 M1
1.6
1.2
0.8
0.4
0.0
2.0
M2 M2
1.6
1.2
0.8
0.4
0.0
2.0
M3 M3
1.6
1.2
0.8
0.4
0.0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)
Figure 5a. Malondialdehyde (MDA) content of Zibdia (A) and Hindi (B) cultivars harvested in
different maturity stages: immature (M1), half-mature (M2) and full mature fruits (M3), measured of
fruit peel stored at versus storage time at 5 different storage temperatures. Symbols represent the
- 4, -▲- 7, -¡- 10 and -«-200C). MDA content expressed as mean (n=3). The vertical bars indicated
to L.S.D. at p=0.05 for two period (0- up to 20 days) when all treatments were present and the
second is from (0- up to 35 days) for all treatments except for storage at 20oC.
176
PULP
2.0
ZIBDIA HINDI
M1 M1
1.6
1.2
0.8
0.4
0.0
2.0
M2 M2
1.6
1.2
0.8
0.4
0.0
2.0
M3 M3
1.6
1.2
0.8
0.4
0.0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Storage time (days)

Figure 5b. Malondialdehyde (MDA) content of Zibdia and Hindi varieties harvested in different
maturity stages: immature (M1), half-mature (M2) and full mature fruits (M3), measured of fruit pulp
stored versus storage time at 5 different storage temperatures. Symbols represent the storage
temperatures are (Zibdia: -{- 2, -- 4, -U- 7, -- 10 and -«- 200C) and (Hindi: -z- 2, - - 4, -▲-
7, -¡- 10 and -«-200C). MDA content expressed as mean (n=3). The vertical bars indicated to
177
Chapter 6
The activity of ACC synthase is directly proportional to the storage temperature

according to Kader (2002). This is shown for both varieties at day 5, and especially
for Zibdia (Figure 1). The near zero level of ethylene production seen after 25
storage days when fruits were stored at 2 and 4oC has been attributed to a lower
activity of ACC synthase at these temperatures and storage times
A difference in the ethylene production rates among the different maturity
stages was also observed: that M3 fruits produce ethylene at a higher rate
compared to M1 and M2 fruits. The same result was reported by Kader (2002) and
Majeed and Jeffery (2002). Since all the maturity stages show the same initial
ethylene production rate, which thereafter changes according to the storage
temperature and the maturity stage, it can be suggested that the activity of ACC
synthase enzymes in forming ethylene is initiated only after fruit harvest (5 days).
The results above can be used to study correlations between ethylene evolution
and lipid peroxidation (Figure 6). The severity of chilling injury symptoms developed
after 20 days of storage were reported in chapters 3 and 4. The development of
oxidative damage in relation to ethylene differs between the peel and pulp.
Ethylene evolution reaches a maximum after 5 days of storage, and for the fruits
stored at 20°C when a sharp increase in the formation of MDA equivalents develop
in the peel (i.e. Zibdia M2 and M3, and Hindi M3 fruits) it does so after 5 days; so it
is fair to argue that in these cases the onset of MDA equivalent formation is
correlated with the C2H4 maximum. In the other maturity stages that do not show
the sharp increase in formation of MDA equivalents in the peel samples, it is also
reasonable to argue that though the fruits are producing ethylene, oxidative
processes leading to membrane breakdown do not become sensitive to ethylene; if
harvested in an immature phase the fruits never develop this sensitivity to C2H4.
The change in concentration of MDA equivalents in the pulp at 20°C in relation to
changes in C2H4 evolution differs to that of the peel, indicating that there is tissue
specificity as a well as maturity dependency in the C2H4/oxidation relationship. The
formation of MDA equivalents in the pulp does not show the dramatic increase that
is evident in some of the peel samples. Rather the increase is more progressive
and begins from the day 0 of storage, though some samples (M1 and M2 of Zibdia,
M1 of Hindi) do show an increase of production after day 10 of storage. Under low
temperature storage the formation of MDA equivalents could be influenced by both
chilling and C2H4. Relative to the 20°C storage the rise in the concentration of MDA
equivalents is slower at chilling temperatures, and is increased by decreasing
temperatures.
Considering firstly processes in the peel, the increase in MDA equivalents at
low temperatures for Zibdia M2 and M3 and Hindi M3 differs markedly from that at
20°C, whereas MDA equivalents formation in Hindi M1 and to a lesser extent M2 is
similar to that at 20°C.
178
PEEL
2.0 ZIBDIA HINDI
y = -0.0027x + 0.5658
o y = -0.0036x + 0.5018 o
1.6 2 C R 2 = 0.4193 2 C R 2 = 0.3823
y = -0.0027x + 0.6069
y = -0.005x + 0.6254 R 2 = 0.6581
1.2 R 2 = 0.8095
y = -0.0032x + 0.6486
0.8 y = -0.0055x + 0.6897 R 2 = 0.8427
R 2 = 0.8751
0.4
0.0
2.0 y = -0.007x + 0.6973 y = -0.0056x + 0.7381
o o
4 C R 2 = 0.6417 4 C R 2 = 0.4888
1.6
y = -0.0105x + 0.9056 y = -0.007x + 0.8602
R 2 = 0.6018 R 2 = 0.4945
1.2
y = -0.0105x + 0.977 y = -0.0084x + 0.9819
0.8 R 2 = 0.5975 R 2 = 0.5727
0.4
0.0
2.0 y = -0.0066x + 0.6632
o y = -0.0071x + 0.8475
7 C o
MDA content peel (ηM g-1 FW)
R 2 = 0.51
1.6 7 C R 2 = 0.6676
y = -0.0073x + 0.7544 y = -0.0078x + 0.9273
R 2 = 0.6399
1.2 R 2 = 0.6077
y = -0.0113x + 0.9994 y = -0.0118x + 1.2554

0.8 R 2 = 0.637 R 2 = 0.5873
0.4
0.0
2.0
y = -0.0086x + 0.9847
o y = -0.0102x + 0.8517 o
1.6 10 C R 2 = 0.7255 10 C R 2 = 0.6389
y = -0.0094x + 1.0998
y = -0.0108x + 0.9303
R 2 = 0.6692
1.2 R 2 = 0.5657
y = -0.0099x + 1.1721
y = -0.0095x + 0.9204 R 2 = 0.6186
0.8
R 2 = 0.4389
0.4
0.0
2.0
y = -0.0095x + 0.9334
o o y = -0.0063x + 0.8691
1.6 20 C R 2 = 0.8208 20 C R 2 = 0.8615
1.2 y = -0.0123x + 1.2549 y = -0.0065x + 0.9397

R 2 = 0.981 R 2 = 0.9031
0.8
0.4 y = -0.0176x + 1.609 y = -0.0165x + 1.6794

R2 = 0.9521 R 2 = 0.9806
0.0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 90
Ethylene production (ml kg-1h-1)
Figure 6a. The linear relationship of malondialdehyde (MDA) content of fruit peel in function of
ethylene production rate on fruits of Zibdia and Hindi varieties harvested in different maturity stages
different storage temperatures (2, 4, 7, 10 and 20oC), over 35 days.
179
Chapter 6
PULP
2.0
ZIBDIA HINDI
y = -0.0065x + 0.5935 y = -0.0042x + 0.6888
o o
2 C R2 = 0.7503 2 C R2 = 0.8094
1.6
y = -0.0053x + 0.6567
1.2 R2 = 0.6465 y = -0.0043x + 0.8146
y = -0.0066x + 0.7869 R2 = 0.5119
0.8 R2 = 0.6326
0.4 y = -0.0052x + 0.9563

R2 = 0.4407
0.0
2.0 y = -0.0105x + 0.8005 y = -0.0054x + 0.724
1.6
4oC R2 = 0.6684 4 oC R2 = 0.5022
y = -0.0121x + 0.9983 y = -0.007x + 0.9262
R2 = 0.5724 R2 = 0.5187
1.2
y = -0.0149x + 1.4142 y = -0.0079x + 1.0685
0.8 R2 = 0.8991 R2 = 0.5898
0.4
0.0
2.0
7 oC y = -0.0112x + 0.8353
7 oC
y = -0.011x + 1.1218
MDA content pulp (ηM g-1 FW)
R2 = 0.5947
1.6 R2 = 0.5223
y = -0.0128x + 1.0723
1.2 R2 = 0.5118 y = -0.0101x + 1.1763
R2 = 0.5398
y = -0.0141x + 1.26
0.8
R2 = 0.7697
0.4 y = -0.0096x + 1.3001
R2 = 0.544
0.0
2.0 y = -0.0095x + 0.7851 y = -0.0075x + 0.8994
10oC R2 = 0.8903 10oC R2 = 0.6407
1.6
y = -0.009x + 0.8774
R2 = 0.7244 y = -0.0068x + 1.1119
1.2 R2 = 0.6004
y = -0.009x + 0.9756
0.8 R2 = 0.5173
0.4 y = -0.0079x + 1.276

R2 = 0.7289
0.0
2.0 y = -0.0045x + 0.5672 y = -0.0049x + 0.8425
20oC R2 = 0.7002 20oC R2 = 0.7962
1.6 y = -0.0036x + 0.6239
R2 = 0.8247 y = -0.0037x + 0.8771
1.2
y = -0.0031x + 0.671 R2 = 0.7823
0.8 R2 = 0.5992
0.4 y = -0.0041x + 0.9831

R2 = 0.6185
0.0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 90
Ethylene production (ml kg-1h-1)
Figure 6b. The linear relationship of malondialdehyde (MDA) content of fruit pulp in function of
ethylene production rate on fruits of Zibdia and Hindi varieties harvested in different maturity stages
180
These responses do not have any strong correlation with changes in C2H4
formation; plotting the MDA equivalent content against C2H4 formation (Figure 6)
reveals that, overall, as C2H4 evolution decreases then MDA content increases, but
that in detail the increase in MDA content can be seen to increase strongly when
the rate of C2H4 evolution has fallen to low values (e.g. Figure 1, 4°C data). In the
pulp under low temperature storage there is also no clear correlation between C2H4
formation and MDA formation. For example the large variation in C2H4 formation
that occurs in the M3 stages of both Zibdia and Hindi is not paralleled by similar
variations in MDA concentration. Nor is there for the pulp at low temperatures any
evident temporal correlation between C2H4 formation and the increase in MDA
concentration. The conclusion is therefore that though C2H4 may be correlated with
increases in membrane oxidation in the peel at 20°C at more advanced maturity
stages, there is no clear correlation at low temperatures in the peel, or for the pulp
at any temperature. Note, however, that the absence of a correlation does not rule
out an indirect causality. Another conclusion that can be drawn from the low
temperature data is that though it is possible for C2H4 to be formed as an oxidized
fatty acid breakdown product, the decrease of C2H4 evolution with increasing MDA,
and the independence of C2H4 evolution shown when C2H4 evolution is low and
MDA levels are increasing (Figure 5), indicates that compared to biochemical
formation of C2H4 the non-enzymic production by membrane oxidation is
insignificant.
4.2. Respiration rate (CO2)
Respiration is the process by which stored organic materials (Carbohydrates,

proteins and fats) are broken down into simple products such as carbon dioxide
and water vapour and accompanied by evolution of energy (Kader, 2002; Noodén
and Leopold, 1988). The loss of stored food reserves in the fruit through respiration
means the hastening of senescence. As the reserves that provide energy to
maintain living status are exhausted, food value for the consumer is reduced
through loss of flavor quality and loss of salable dry weight. The rate of
deterioration of harvest commodities is generally proportion to the respiration rate.
Horticultural crops can be classified according to their respiration rates and mango
fruits are classified as moderate respiration (Kader, 2002). Several studies have
reported that ethylene activates respiratory enzymes in different processes in
different fruits. For example, ethylene treatment increases the activity of malic
enzymes in climacteric fruits (pome fruits) and the respiration of avocado and
banana in the pre-climacteric stage, as reviewed by Noodén and Leopold (1988).
Mango fruits undergo an initial climacteric respiration, reaching a maximum at day
181
Chapter 6
10 of storage at all maturity stages (Figure 2). This is five days later than the
maximum of C2H4 evolution, and this result differs from previous reports.
The increased respiration rates of Zibdia and Hindi mango varieties occurred at day
10 of this experiment (after 5 days of the ethylene peak). A similar pattern was
described previously for the ‘Kent’ mango variety, but a reversed pattern was
observed in the ‘Haden’ variety (Trinindad et al., 1997). The variation of the order of
respiration and ethylene production after harvesting indicates that ethylene and
respiration production depend on mango varieties which (McCollum et al., 1993). It
is suggested that in our case, ethylene production is an important factor in the
activation of respiratory enzymes. The observed climacteric burst at day 10 of
storage may be caused by enhancement of the phosphorylation process,
increasing concentration of ATP as an essential energy molecule for the chemical
reactions of respiration (Noodén and Leopold, 1988). This peek is very temperature
sensitive (Figure 2). After the climacteric peak, respiration rate decreased rapidly to
very low levels after 15 days of storage. This low level is maintained up to 35 days
but with a slight increase with increasing storage time in all maturity stages of both
mango varieties. These increases of respiration were found towards the end of the
experiment (days 20-35). This is in accordance to the previous results by Dinora et
al. (1997). Increased CO2 production by increasing maturity of fruit was also
observed in ‘Tommy Atkins’ by Majeed and Jeffery (2002). We found a positive
relationship between MDA and respiration in both mango varieties (Figure 6).
However, the correlation was generally above 50% in the cold stored mango
whereas it and was much less in the case of mango peel stored at 20oC for both
varieties. The relation was more significant when storage temperature was lowered
(2oC to 10oC) compared with 20oC for fruit pulp. These patterns shown suggest that
respiration may affect peroxidation of lipid cell membrane indirectly, by forming
AOS during respiration processes (Meir, 1986). It would be also be that as the rise
of MDA indicates increase membrane damage, some uncoupling of respiration may
have occurred. Enzymes and non-enzyme antioxidants declined until the end of the
experiment as described in chapters 4 and 5.
4.3. Firmness
Firmness is one of the most important product attributes determining product
acceptability to the consumer. As an attribute, firmness depends on many
properties of the tissue: water content, the nature of the cell wall and turgor are
clearly important sources of firmness, but the cell contents can also play a part.
Changes in firmness should, in principle, be traceable to alterations in the various
tissue components or properties that combine to create the firmness attribute, and
PEEL
182
ZIBDIA HINDI
2.0
o y = 0.0094x + 0.3564
o
2 C R2 = 0.3804 2 C
y = 0.0086x + 0.4426
1.6 R 2 = 0.3997
1.2 y = 0.0052x + 0.4832 y = 0.0089x + 0.4615

R2 = 0.1095 R 2 = 0.5121
0.8
0.4 y = 0.0046x + 0.519 y = 0.0055x + 0.5066
R2 = 0.083 R 2 = 0.2487
0.0
2.0
o y = 0.0224x + 0.4563
4 C y = 0.0114x + 0.4473 o
4 C R 2 = 0.3978
1.6 R 2 = 0.4669
1.2 y = 0.0306x + 0.3132 y = 0.0345x + 0.3593

R 2 = 0.7021 R 2 = 0.6482
0.8
0.4 y = 0.0208x + 0.456

y = 0.0355x + 0.3753
R 2 = 0.6434
R 2 = 0.517
0.0
2.0
y = 0.0171x + 0.3255 o y = 0.0141x + 0.4959
1.6 7 oC R2 = 0.7617 7 C R 2 = 0.3287
1.2 y = 0.0161x + 0.3777

y = 0.0203x + 0.4663
R2 = 0.6379
R 2 = 0.524
0.8
0.4 y = 0.0228x + 0.3788 y = 0.025x + 0.5616

R2 = 0.5488 R 2 = 0.3844
0.0
2.0
y = 0.0174x +0.36 y = 0.0213x + 0.4549
o o
1.6 10 C R2 = 0.6448 10 C R 2 = 0.6978
1.2
y = 0.0203x +0.3246 y = 0.0218x + 0.4802
0.8 R2 = 0.8059 R 2 = 0.7054
0.4 y = 0.0159x +0.4223 y = 0.0184x + 0.5524

R2 = 0.6297 R 2 = 0.6045
0.0
2.0
y = 0.0006x + 0.6349
20 C
o y = -0.0025x + 0.6179 20oC R2 = 0.0024
1.6 R2 = 0.0263
y = 0.0004x + 0.6944
1.2 y = -0.0042x + 0.8153 R2 = 0.0027
R2 = 0.085
0.8
0.4 y = -0.0027x + 1.0092 y = -0.0036x + 1.1014

R2 = 0.0773 R2 = 0.0506
0.0
0 30 60 90 120 150 180 0 20 40 60 80 100
Respiration rate (CO2 mg h-1 kg-1)
respiration rate of fruits of Zibdia and Hindi varieties harvested in different maturity stages (Zibdia: -
{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3), which stored at different
storage temperatures (2, 4, 7, 10 and 20oC), over 35 days.
PULP
183
Chapter 6
ZIBDIA HINDI
2.0 o y = 0.0076x + 0.5477
2 C y = 0.0117x + 0.383 2 oC R2 = 0.2648
1.6 R2 = 0.3285
y = 0.0173x + 0.5496
1.2 y = 0.0071x + 0.4888 R2 = 0.5835
R2 = 0.147
0.8
0.4 y = 0.0101x + 0.5252
y = 0.012x + 0.6858
R2 = 0.2036
R2 = 0.2328
0.0
2.0
4oC y = 0.0174x + 0.4227 y = 0.021x + 0.4565
1.6 R2 = 0.5085 4 oC R2 = 0.3725
1.2 y = 0.0354x + 0.4169

y = 0.0355x + 0.3112
R2 = 0.6697 R2 = 0.7221
0.8
0.4 y = 0.02x + 0.8213 y = 0.0309x + 0.5275
R2 = 0.3566 R2 = 0.5671
0.0
2.0 y = 0.0227x + 0.5665
1.6 7 oC
y = 0.0295x + 0.2534
R2 = 0.7932
7 oC R2 = 0.3192
1.2 y = 0.0331x + 0.351

y = 0.0278x + 0.5574
2 R2 = 0.5184
R = 0.7106
0.8
y = 0.0252x + 0.5343
0.4 y = 0.0225x + 0.7003
R2 = 0.5206 R2 = 0.4312
0.0
2.0
y = 0.02x + 0.4154
10oC
y = 0.0148x + 0.3473
1.6 R2 = 0.6615 10oC R2 = 0.7905
1.2 y = 0.0139x + 0.4208 y = 0.016x + 0.6625

R2 = 0.6889 R2 = 0.656
0.8
0.4 y = 0.0142x + 0.5215 y = 0.0124x + 0.8245

R2 = 0.6519 R2 = 0.5085
0.0
2.0 y = 0.0003x + 0.6633
y = -0.0004x + 0.4081 o
20 C o 2
R = 0.0023
20 C R 2 = 0.001
1.6 y = -2E-06x + 0.7402
R 2 = 3E-07
y = -0.0013x + 0.4978
1.2
R2 = 0.0818
0.8
y = -0.0007x + 0.8327
0.4 R 2 = 0.0174
y = -0.001x + 0.585
R2 = 0.2336
0.0
0 30 60 90 120 150 180 0 20 40 60 80 100
Respiration rate (CO2 mg h-1kg-1)
respiration rate of fruits of Zibdia and Hindi varieties harvested in different maturity stages (Zibdia: -
{- M1, -- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3), which stored at different
storage temperatures (2, 4, 7, 10 and 20oC), over 35 days.
184
storage conditions should be chosen to minimize alterations in these components

and properties (Sams, 1999).
The mechanical nature of firmness has lead to attention being directed
towards cell wall changes. (Ketsa et al., 1999b; Watada et al.,1984). The
breakdown or modification of cell wall polysaccharides during the ripening of
mango fruits is associated with changes in firmness, and these changes are the
result of the increased activity of polygalacturonases, cellulase and β-galactosidase
(Ali et al., 1995; Huber, 1983, Ketsa et al., 1999b). Based on these, an explanation
could be suggested for the rapid decrease in firmness of Zibdia and Hindi fruits
when they are stored at 20oC for 20 days. During this period, hydrolase enzymes
may act on the cell wall resulting in the rapid decrease of fruit firmness as shown in
Figure 3. In connection, these chemical changes can be associated with increased
C2H4 and respiration during storage of fruits at 20oC by which the cell wall
hydrolysis is enhanced. This is clear from our results that the decrease in fruits
firmness after day 5 of storage at 20OC (Saltveit, 1989). It is clear that the metabolic
interaction between ethylene synthesis, respiration and decrease in fruit firmness
may be attributed to increases of AOS levels during these processes. The increase
in oxidation is coupled with a decline in antioxidant enzyme activity (see chapter 5),
which eliminate AOS formation during the ripening processes (Aharoni et al., 2002).
These responses may be stimulated by AOS formation during cell membrane
peroxidation, further accelerating the ripening and senescence processes (Hodges,
2003).
Decreasing firmness is closely related to increasing amounts of MDA in the
peel and pulp of both fruits (Figure 5). This relationship is consistent in both
cultivars and across the three maturity stages under conditions of low temperature
storage. At 20°C the relationship between MDA equivalents and firmness remains
to only a limited degree for the peel, but has disappeared for the pulp. This
suggests a role for oxidative stress in modulating the loss of firmness under low
temperature conditions, but to a much lesser degree under 20°C storage. More
broadly, it may the under low temperature storage the ripening and senescence
processes are modulated by oxidation. By accelerating the ripening process AOS
can lead to firmness changes via increases in enzymatic cell wall hydrolysis. In
addition, however, AOS can directly break down the cell wall (Hodges 2003). They
may also reduce firmness by damaging the cell membrane, and reducing turgor
formation: turgor requires both a cell wall and a cell membrane, and though much
attention is directed to the role of the cell wall, the membrane may also play a part.
Indeed, one interpretation of the relationship between firmness and the
accumulation of MDA (Figure 7) equivalents is that the loss of firmness is due to
oxidative membrane damage, rather than a more general AOS induced
185
Chapter 6
senescence. Fruit firmness is correlated not only with the accumulation of MDA
equivalents, but also with increasing ion leakage (Figure 8) from both peel and pulp
samples for both cultivars and over all temperatures and maturity stages.
Membrane leakiness would be expected to be associated with a reduction of turgor
and firmness, and oxidative damage to the membrane will increase leakiness.
However our data do not exclude the possibility that membrane leakiness is a
consequence of cell wall breakdown and not a cause of loss of firmness.
Acknowledgements
The author was supported by a grant from the Egyptian High Educational
Ministry for full program.
186
PEEL
2.0 ZIBDIA HINDI
y = -0.0119x + 1.6575
o
1.6
2 C R2 = 0.7841 o
2 C
y = -0.0031x + 0.8851
R 2 = 0.8467
y = -0.0021x + 0.7589
1.2 y = -0.0096x + 1.4377 R 2 = 0.7854
2
R = 0.8702 y = -0.0046x + 1.0591
0.8 R 2 = 0.9173
0.4 y = -0.0065x
2
+ 1.2182
R = 0.8423
0.0
2.0 y = -0.0053x + 1.2034 y = -0.0053x + 1.2195
4oC R2 = 0.7576
o
4 C R2 = 0.7638
1.6
y = -0.0133x + 2.2711
1.2 y = -0.0077x + 1.4422
2 R2 = 0.8064
R = 0.824
0.8
0.4 y = -0.0095x + 1.5205 y = -0.0146x + 2.2827
R2 = 0.6294 R2 = 0.6459
0.0
2.0 y = -0.0082x + 1.6542
7 oC
o y = -0.0077x + 1.2103
1.6 7 C R2 = 0.883 R2 = 0.6678
y = -0.01x + 1.8899
1.2 y = -0.0109x + 1.5772 R2 = 0.6357
R2 = 0.8881
0.8
0.4 y = -0.0106x + 1.5897 y = -0.0212x + 3.1531
R2 = 0.5315 R2 = 0.7193
0.0
2.0 y = -0.0039x + 0.9683
o
1.6
10 C y = -0.0059x + 1.0462
2
10oC R2 = 0.5835
R = 0.7195
y = -0.0057x + 1.3074
1.2 y = -0.0079x + 1.3062 R2 = 0.8243
2
R = 0.8282
0.8
0.4 y = -0.008x + 1.2403 y = -0.0067x + 1.2551
R2 = 0.6097 R2 = 0.6204
0.0
2.0 y = -0.0034x + 0.9963
20oC y = -0.0055x + 0.8829
20oC R2 = 0.6805
1.6 R2 = 0.6702
y = -0.0044x + 1.0379
1.2
R2 = 0.8556
y = -0.0254x + 3.2716
0.8 R2 = 0.4826
0.4 y = -0.0129x + 2.1632

y = -0.0092x + 1.5691
R2 = 0.7759
R2 = 0.5431
0.0
0 30 60 90 120 150 180 0 30 60 90 120 150 180
Fruit firmness (N)
firmness of fruits of Zibdia and Hindi varieties harvested in different maturity stages (Zibdia: -{- M1,
-- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3), which stored at different storage
temperatures (2, 4, 7, 10 and 20oC), over 35 days.
187
Chapter 6
PULP
ZIBDIA HINDI
2.0 y = -0.0166x + 2.1768 y = -0.0033x + 1.0003
2 oC R2 = 0.848 2oC R2 = 0.7899
1.6
1.2 y = -0.0114x + 1.6349 y = -0.0039x + 1.1057
R2 = 0.8839 R2 = 0.8086
0.8
0.4 y = -0.0089x + 1.5299 y = -0.0098x + 1.8602
R2 = 0.8066 R2 = 0.8143
0.0
2.0 y = -0.0053x + 1.2044
y = -0.0082x + 1.5859 o
o 4 C R2 = 0.811
1.6 4 C R2 = 0.8403
y = -0.0131x + 2.3152
y = -0.0089x + 1.6156
MDA content pulp (ηM g-1 FW)
1.2 R2 = 0.8296
R2 = 0.7759
0.8
0.4 y = -0.0123x + 2.0991 y = -0.0128x + 2.1974
R2 = 0.801 R2 = 0.5794
0.0
2.0 y = -0.013x + 2.4034
o y = -0.013x + 1.752
7 C R2 = 0.8716
o
7 C R 2 = 0.6226
1.6
y = -0.0134x + 2.4678
1.2 y = -0.0214x + 2.7325 R 2 = 0.5962
R2 = 0.9051
0.8
0.4 y = -0.0133x + 2.0057 y = -0.0171x + 2.8293
R2 = 0.6522 R 2 = 0.6507
0.0
2.0
10oC o y = -0.0035x + 0.8839
1.6
y = -0.0048x + 0.9171 10 C R2 = 0.5792
R2 = 0.6944
y = -0.0041x + 1.2668
y = -0.0058x + 1.1279
1.2 R2 = 0.7647
R2 = 0.8289
0.8
0.4 y = -0.0074x + 1.2668 y = -0.0053x + 1.3423
R2 = 0.6838 R2 = 0.7333
0.0
2.0
o
20oC
y = -0.0028x + 0.5548
R2 = 0.6686
20 C y = -0.0026x + 0.9387
1.6 R 2 = 0.6198
y = -0.0091x + 1.3777
1.2 R2 = 0.6129 y = -0.0026x + 0.9378
y = -0.003x + 0.8432 R 2 = 0.7747
0.8 R2 = 0.6153
0.4 y = -0.0024x + 0.9586

R 2 = 0.5122
0.0
0 30 60 90 120 150 180 0 30 60 90 120 150 180
Fruit firmness (N)
firmness of fruits of Zibdia and Hindi varieties harvested in different maturity stages (Zibdia: -{- M1,
-- M2 and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3), which stored at different storage
188
PEEL
100
ZIBDIA HINIDI
y = -1.1049x + 145.43 o y = -0.4515x + 90.489
o 2C
80 2 C R 2 = 0.7571 R 2 = 0.7565
y = -0.5124x + 88.58
60 y = -0.7167x + 108.53 R 2 = 0.7209
R 2 = 0.6999
40
20 y = -1.4832x + 178.55 y = -0.3675x + 72.28
R 2 = 0.7055 R 2 = 0.6933
0
100
o y = -0.3807x + 83.622
80 4 C R 2 = 0.7153 4 C
o y = -0.6995x + 118.85
R 2 = 0.5883
y = -0.9148x + 131.32
60 R 2 = 0.6503 y = -1.0727x + 151.13
R 2 = 0.5091
40
20 y = -0.4714x + 83.433 y = -1.2114x + 178.92

R 2 = 0.7834 R 2 = 0.701
0
100
o o y = -0.7574x + 131.15
80 7 C y = -1.0649x + 141.85
R 2 = 0.7402
7 C R 2 = 0.411
60 y = -1.5176x + 184.35
y = -0.6103x + 104.53
R 2 = 0.8183
R 2 = 0.552
40
20 y = -0.7165x + 106.72 y = -1.1684x + 167.84

R 2 = 0.376 R 2 = 0.6526
0
100
o y = -0.3192x + 64.008
10 C y = -0.4062x + 74.523 o
10 C R 2 = 0.4349
80 R 2 = 0.5776
y = -0.431x + 78.372 y = -0.4708x + 88.34

60 R 2 = 0.7622 R 2 = 0.8896
40
20 y = -0.4019x + 67.625 y = -0.2696x + 55.9

R 2 = 0.6114 R 2 = 0.4883
0
100
o y = -0.3999x + 66.399 o y = -0.3731x + 82.155
20 C R 2 = 0.7644 20 C R 2 = 0.4993
80
60 y = -1.1334x + 156.34 y = -0.4135x + 74.083

R 2 = 0.5026 R 2 = 0.6737
40
20 y = -0.5531x + 101.93 y = -0.2806x + 60.201

R 2 = 0.6553 R 2 = 0.591
0
0 40 80 120 160 200 0 40 80 120 160 200
Fruits firmness (N)
Figure 8a. The linear relationship of malondialdehyde (MDA) content of fruit peel in function of fruits
firmness of Zibdia and Hindi varieties harvested in different maturity stages (Zibdia: -{- M1, -- M2
and -U- M3) and (Hindi: - - M1, - - M2, and -▲- M3), which stored at different storage
189
Chapter 6
PULP
100
ZIBDIA HINDI
y = -0.0033x + 1.0003
o y = -2.1923x + 273.14 o
80
2 C R 2 = 0.667
2 C R 2 = 0.7899
y = -0.0039x + 1.1057
60 y = -1.4778x + 191.32 R 2 = 0.8086
R 2 = 0.6704
40
20 y = -0.772x + 124.61
y = -0.0098x + 1.8602
R 2 = 0.6417 R 2 = 0.8143
0
100
o y = -0.7181x + 118.89 y = -0.0053x + 1.2044
o
4 C R 2 = 0.6357 4 C R 2 = 0.811
80 y = -0.0131x + 2.3152
y = -0.49x + 105.64 R 2 = 0.8296
60 R 2 = 0.763
40
20 y = -0.525x + 100.67 y = -0.0128x + 2.1974

R 2 = 0.6884 R 2 = 0.5794
0
100
y = -0.013x + 2.4034
o y = -0.7936x + 122.68 o
7 C R 2 = 0.8395 7 C R 2 = 0.6226
80
y = -0.0134x + 2.4678
y = -0.5846x + 100.36 R 2 = 0.5962
60
R 2 = 0.853
40
20 y = -0.5845x + 102.25 y = -0.0171x + 2.8293

R 2 = 0.355 R 2 = 0.6507
0
100
o y = -0.3627x + 80.398 y = -0.0035x + 0.8839
10 C R 2 = 0.6655 10oC R2 = 0.5792
80
y = -0.5039x + 98.793 y = -0.0041x + 1.2668
60 R 2 = 0.7852 R2 = 0.7647
40
y = -0.3485x + 74.45
20 y = -0.0053x + 1.3423
R 2 = 0.5855
R2 = 0.7333
0
100
y = -0.3255x + 74.615 y = -0.0026x + 0.9387
o
20 C R 2 = 0.8246 20 C
o R 2 = 0.6198
80
y = -1.0979x + 170.39 y = -0.0026x + 0.9378
R 2 = 0.7747
60 R 2 = 0.4981
40
20 y = -0.5023x + 106.37 y = -0.0024x + 0.9586

R 2 = 0.5122
R 2 = 0.7314
0
0 40 80 120 160 200 0 30 60 90 120 150 180
Fruits firmness (N)
Figure 8b. Presents the linear relationship of malondialdehyde (MDA) content of fruit pulps in
function of firmness of fruits of Zibdia and Hindi varieties harvested in different maturity stages
190
Chapter 7
Final comments
Chapter 7
Perhaps unsurprisingly, the results presented in this thesis indicate that Mango
fruits are indeed chilling sensitive, which is entirely in accordance with the known
physiology of this product. The detail offered in this study, however, reveals many
new features of the response of these fruits to chilling. One of the most surprising
was the extent to which organized metabolism could continue in these tropical
products even at 2°C. Both protein and metabolite synthesis were shown to be
active at this temperature, which confounds the usual impression that tropical
plants products at such temperatures are at best essentially metabolically inactive.
The greater damage that often occurred at 4°C compared to 2°C was also
unexpected. This implies that chilling injury, though dependent on low
temperatures, also has an activation energy and that it therefore also has a positive
temperature dependence. Coupled with the previous comments about continued
metabolic activity, the possibility remains that chilling injury in these fruits has a
metabolic component.
As far the detailed properties of chilling sensitivity go, it is clear that storage
at 10°C or possibly even 7°C is feasible, at least as far the physiological status of
the fruits is concerned. At 10°C temperature the development of chilling related
injuries was least severe yet there was some low temperature induced suppression
fruit quality due to ripening. However suitability for 10°C is cultivar dependent. For
example, the progress of fruit softening in both Hindi and Zibdia depended on both
cultivar and maturity stage; in Zibdia the M1 fruits showed slower softening at 10°C
compared to 20°C, whereas in Hindi 10°C storage did not retard softening, except
in the M2 fruits. In any case 10°C storage gave at most 15 days before softening
commenced. However, though in Hindi, though not greatly retarded by storage at
10°C, was markedly retarded by storage at lower temperatures (eg 7°C). These
lower temperatures, however, though retarding the loss of fruit firmness,
accelerated the development of chilling induced injuries. At 7°C storage, the CI-
index began to increase from 1 (the value representing no damage) after the 20th
day of measurement. Ion leakage and equivalents also increased more at 7°C than
10°C, though the difference was only slight. The ascorbic acid and tocopherol
content of the pulp also only decreased slightly more at 7°C storage compared to
10°C, though the rise in carotene levels was conspicuously lower at 7°C. So,
limited low temperature storage is possible, especially if attention is paid to cultivar
selection, and certainly storage at 10 - 7°C, which seems realistic in physiological
terms, is better than storage at 20°C
With regards to storage the future use of the fruit is a major that should
guide the choice of storage conditions and duration of storage. The levels of
ascorbic acid and tocopherol were decreasing from the first day of measurement,
and in the case of ascorbic acid, levels could decrease to nearly zero. In the case
192
Final comments
of these vitamins (C and E) any use of for which a high vitamin content was
important would require immediate processing. Carotene, on the other hand,
increased during the storage period, so for high carotenoid levels, storage is
benefical, though processing or use would need to be synchronized with the
maximum of carotenoid production, as carotenoid level decrease rapidly oncd they
have reached a maximum. Genetic and tissue variability in the initial quantities and
rate of change of content of components (eg vitamin content and change) and
properties ( firmness) is apparent even in this limited study of two cultivars. This
suggests that more purposeful breeding of cultivars for more clearly defined
purposes (eg storage, high carotene content, high vitamin C content) would be a
possible means by which the usefulness and quality of mango fruits could be
improved.
With respect to the origins of chilling injury in a shift in the balance between
oxidative processes and anti-oxidative mechanisms, the number of different
properties measured allows more insight to be gained as to the mechanisms that
seem to be important in causing or preventing oxidative injury to the tissues of
mango fruits. A major problem with the interpretation of the type of data available is
its intrinsic complexity. Oxidative and anti-oxidative processes exist in a network
rather than in linear sequences of reactions or events. Such networks are
fundamentally more difficult to analyse in terms of limiting steps or bottlenecks. We
have not had sufficient control over the mango system to allow it to be manipulated
in a way that would allow a more sensitive analysis of flux control or causality.
Nonetheless, some relationships were conspicuously strong, which suggests that
would be worth exploring in more detail. The amounts of both ascorbic acid and
tocopherol seem to correlate well and inversely with various indices of damage;
MDA formation and CI-index. Especially with MDA accumulation and ion leakage
the relationships were consistent across the two varieties. Ion leakage and MDA
accumulation were also closely related; this is a particularly intriguing correlation
because of the debate concerning the relative roles of membrane dysfunction and
oxidative damage as the primary cause of injury. These data show that changes in
antioxidant status are closely coupled in time to increases in lipid oxidation and ion
leakage. This could be interpreted as indicating that the processes of oxidation and
membrane stability changes are tightly linked and interdependent. The close
relationship between ion leakage and membrane oxidation is especially important
as it illustrates graphically the importance of protecting the membrane against
oxidation.
The antioxidant metabolites exist in a network with antioxidant enzymes. The
relative important of different enzymes in different contexts is rarely explored in
depth, however the data presented here does allow something to be said about the
193
Chapter 7
relative importance of different enzymes in relation to protection against different

injuries. Considering ion leakage, MDA accumulation and protein carbonyl levels as
indices of damage, it is clear that the intensity of damage was not correlated to
SOD or GR activities. These indices of damage were better correlated with the
activities of APX and CAT, though the correlations were not as homogeneous as
those encountered when the concentrations of ascorbic acid or tocopherol were
compared against indices of damage. They were typically more dependent on
maturity stage, for example. Nonetheless, it is suggested that the activities of CAT
and APX and their role in protecting against damage should be further investigated.
Finally regarding the consequences of damage, it is interesting that a possible role
has been identified for membrane damage and leakiness in the loss of firmness
that develops during storage at low temperatures. Future research should establish
the relative importance of cell wall and cell membrane changes with regards the
softening of tissue during ripening and chilling injury.
The last comments concern the measurement of chilling injury. Though
fluorescence has been widely used as a tool with which to measure ripening or
injury, it appears that quantitatively the Fv/Fm parameter, at least as far as mango
fruits under chilling conditions are concerned, does not correlate very well with
other indices of damage, such as electrolyte leakage, chlorophyll content or CI-
index. When measured from fruits that have been subjected to cold storage
conditions, these indices correlate well with each other, so they appear to
consistent indices with which to measure chilling injury. However, qualitatively the
fluorescence Fv/Fm parameter correlates with other indices under chilling
conditions, and the relationship is quantitative for fruits ripening at 20°C. These
results suggest the need to re-examine the use of the Fv/Fm parameter as a tool
for quantifying chilling injury in mango, and possibly to combine it, or even replace
it, with other techniques, such as colour measurements or bioimpedance
measurements.
194
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206
Summary
At present, the value and production quantity of mango fruits are increasing
worldwide. Many studies emphasize how chilling injury phenomena affect the
quality of tropical fruits, such as mango, during postharvest handling, transport and
storage. Since mango is one of the most favored and popular fruits in the world
market, and is considered to be a climacteric fruit, ripening rapidly after harvest, it
is essential to study how storage affects the external and internal fruit quality.
Generally, cold temperatures are used to slow metabolic processes during storage
in order to extend the shelf life. Mango fruit is characterized by short shelf life at
ambient conditions but it is also sensitive to low storage temperatures. Therefore,
the susceptibility and sensitivity of mango fruits to low storage temperatures (ie
below 12oC) coupled with their perishability at non-chilling temperatures limits the
handling, storage and transportation of the fruits. Because of this, the pre and
post-harvest factors involved in the susceptibility of mango fruits to chilling injury
must be carefully studied together to improve shelf life and quality of fruits.
The main aim of this study was to quantify and understand the effects of
storage conditions on the occurrence of chilling injury to mango fruits during long-
term storage at low temperatures. Critical attention has been paid to the
integration of factors that might contribute to susceptibility to injury and attempting
to develop a better understanding of the physiological processes involved with
chilling injury in order to better predict and understand chilling injury. The focus
was on the effect of storage conditions (temperature and duration of storage), on
mango varieties (Zibdia and Hindi Be-Sennara), harvested at different fruit maturity
stages, immature (M1), half-mature (M2) and full mature (M3), with regard to the
appearance of chilling injury symptoms.
Chapter 1 presents an overview of mango as plant, crop, and economic
commodity in both international and local markets.
Physiologically, the effect of low temperatures on plant cells induces
physical and/or physiological disturbances, or injury, in cell membranes and
cytosol Based upon various criteria these injuries can be defined as either primary
or secondary injury. A primary injury is an initial rapid response to low temperature
which causes a dysfunction in the plant cell membranes, metabolic processes, or
cell organisation (e.g. cytoskeleton). This class of injury can be reversed if
temperature is raised to a non-chilling temperature. Long term exposure to low
temperature leads to the development of secondary injury or injuries which are
consequent upon primary injuries. For example in response to chilling cell
Summary
membranes will undergo phase transitions that will result in various dysfunctions,
such as a loss of semi-permeability, incorrect functioning of bound proteins, loss of
specificity of membrane-bound signalling systems etc. In the short term the
consequences of these are reversible, but in the longer term the oxidation of cell
components (eg membranes, proteins) and metabolic disorder is sufficiently
severe that it leads to cell death by necrosis.
Generally, the appearance of chilling injury symptoms is associated with the
occurrence of oxidative stress. The oxidative reactions result in a dysfunction in
cell membranes by generation of AOS, formed by metabolic processes. The most
common AOS forms is O2•− ions and H2O2, which are formed in vivo by a variety of
mechanisms. Some metabolic enzymatic reactions in plant cells (e.g. xanthine
oxidase and urease) will form O2•−.H2O2 may be formed by dismutation of two O2•−
ion, by β-oxidation of fatty acids in the course of lipid metabolism and in response
to stress. Another source of generation AOS is by pathogen infection. Other sites
in plant cells can produce AOS forms, as has been reported in several studies i.e.
in the inner mitochondrial membrane and cell wall, by enzymatic reactions in the
cell membrane. The increasing steady-state levels of AOS during long-term
storage may also be due to the decreasing effectiveness of defence systems
based on enzymatic and nonenzymatic antioxidants. Nonenzymatic antioxidants
include ascorbic acid (ASC), α-tocopherol (α-TOC) and β -carotene (β -CAR).
These molecules are often linked with antioxidant enzymes such as ascorbate
peroxidase, glutathione reductase, which regenerate the antioxidant capacity of
the quenching molecule. Purely enzymatic reactions, such as superoxide
dismutase (SOD), Catalase (CAT), and peroxidase (APX) protect the plant cell by
directly scavenging O2•− and H2O2. The physiological changes are summarised in
Chapter 2.
All experiments were conducted on two Egyptian varieties (Zibdia and Hindi
Be-Sennara) which were used as mango fruit models. Three different maturity
stages at time of harvest, immature, half-mature and fully mature fruits were used
in this work. Storage conditions were set to 5 different temperatures (2, 4, 7, 10
and 20oC) for 35 days. In all the experiments fruit samples were taken at time
intervals of five days. During a range of experiments the effect of chilling was
studied on the water and lipid-soluble antioxidants compounds and anti-oxidant
enzymes,, various chilling injury symptoms (electrolyte leakage, skin chlorophyll
content, chlorophyll fluorescence responses, and a visual injury assessment), and
mechanical properties of the fruits. Where appropriate, both the peel and pulp
portions of the fruits were investigated. The purpose of these studies was to
examine how well different assays of chilling injury worked, how chilling changed
208
Summary
the biochemical properties of the fruits, how these changes correlated with
measurable indices of chilling injury, and finally which to ascertain by means of
correlation studies which antioxidant components appeared most important in
relation to the occurrence of chilling injury.
Chapter 3 describes the possibilities of using chlorophyll fluorescence
parameters (Fv/Fm, F0 and Fm) to assess chilling injury of fruits with the aim of
predicting chilling injury symptoms before they are visible. An attempt has been
made to analyze the effect of storage temperature, conducted on different mango
varieties at different maturity stages. Fruits were stored at 5 temperatures (2, 4, 7,
10 and 20o) for 35 days. Chilling injury index (CI-index) and chlorophyll a, b
content and a/b ratio were measured every 5 days and chlorophyll fluorescence
(CF) every 3 days. It is shown that temperature and variety had a significant effect
with storage time, but no effect of maturity stage was observed. A higher storage
temperature decreased Fv/Fm and Fm, whereas F0 increased during the
experiment. In prolonged storage at low temperature, Fv/Fm decreased more
rapidly up to the end of the experiment in Zibdia than Hindi fruits. In Zibdia, it
decreased after 3 days, compared to 6 days in Hindi, and before visible chilling
injury symptoms appeared. Also, Zibdia presented higher severity of chilling injury
symptoms during storage than Hindi fruits in all maturity stages. CI appeared on
day 10 in both varieties at all maturity stages. It was less severe in M1 fruits than
M2 and M3. Chla degraded more rapidly in Zibdia than Hindi and more rapidly than
Chlb in all maturity stages. It was higher in Zibdia than Hindi. This relationship
allows a prediction of CI before visible symptoms appear. The results show that
Zibdia fruits are more sensitive to low storage temperatures than Hindi fruits.
Therefore, it is necessary to study which factor increases resistance to chilling
temperatures.
The effect of storage temperatures on water and lipid-soluble antioxidants
such as ascorbic acid (ASC), α-tocopherol (α-TOC) and β-carotene (β-CAR)
content during storage and its correlation with CI-index is described in Chapter 4.
The experiment was conducted on two mango varieties ‘Zibdia’ and ‘Hindi’, which
were harvested in three different maturity stages. The fruits were stored at different
storage temperatures, focusing on their response to low temperature, with the aim
of achieving a better understanding of the underlying physiological processes of CI
development with regard to antioxidants which provide a protection against
harmful active oxidative species (AOS). Physiologically, long-term storage
increases the imbalance between the oxidant/antioxidant, which leads to increased
oxidative damage to cell membranes following reactions with membrane lipids and
proteins, which finally results in cell death. Therefore, water and lipid-soluble
antioxidants were measured in fruit components (peel and pulp) during the storage
209
Summary
period. Generally, a higher initial ASC content of both fruits was observed in M1
fruits compared with M2 and M3 fruits. α-TOC and β-CAR showed the opposite
trend. The lowest level of ASC was observed in almost completely damaged fruits.
Rapid decreases in ASC, was observed in half and fully mature fruit and when the
fruits were stored at the low temperature range. A negative linear relationship was
found between water/lipid-soluble antioxidants and CI-index which proves that
water/lipid-soluble antioxidant decreased based on storage temperature, time and
fruit maturity stages.
The data obtained in the previous chapter allows us to study the activities of
antioxidant enzymes (SOD, CAT, APX and GR) and the terminal products of AOS
oxidation of lipids and proteins that occurs during storage. In this experiment,
Zibdia and Hindi fruits were stored at two storage temperatures (4 and 10oC) for 35
days. The results indicate that lipid peroxidation and protein oxidation increase up
to end of the experiment in both varieties and fruit parts. The activity of the
antioxidant enzymes increased to the maximum, dependent on storage
temperature, variety, maturity stage and time. In addition, fruit parts presented
different enzyme activities as to (SOD, CAT, APX and GR) because of storage
temperature effects. A negative linear relationship between MDA content and the
activities of antioxidant enzymes (CAT and APX) was observed. This relationship
shows the imbalance between antioxidant activity and AOS formation, which is
reflected by increasing lipid peroxidation in both mango varieties and fruit parts.
Similarly, a negative linear relationship between protein oxidation (PCG) and
antioxidant enzyme activities (CAT, APX and SOD) was found.
Chapter 5 presents the development of physiological changes such as
ethylene (C2H4) production, respiration rate (CO2), firmness and ion leakage on
mango cultivars. The results indicated that the climacteric respiration peak was
observed on day 10 of storage. In addition, ethylene production reached a
maximum on day 5 of storage. M3 fruits produced the highest amount of C2H4
compared to M1 and M2 fruits in both varieties. The same pattern was observed
with respiration rate. Storage temperature affects C2H4 production and respiration
rate (CO2), which are higher at 20oC than chilling temperatures (2 to 10oC).
Storage at low temperature kept fruit firmness higher in both varieties. Hindi fruits
presented a higher firmness than Zibdia fruits and M1 fruits were firmer than M2
and M3. Another effect of storage temperature observed in this experiment is that
ion leakage increased with lower storage temperatures, which indicates the
dysfunction in the membranes and walls of the plant cell. A relationship between
lipid peroxidation products (MDA) and ethylene production, respiration rate,
firmness and ion leakage was observed. These relationships indicate the
dysfunction of cell membrane due to increased peroxidation processes in the
210
Summary
membrane. MDA had a negative relationship with ethylene (C2H4) production and
fruit firmness. It is clear from the relationships that the storage temperature plays
an important role in increasing lipid peroxidation processes and inhibiting the
hydrolytic enzymes, which are related to fruit softening. On the other hand, a
negative relationship was found between MDA and respiration rate (CO2) and ion
leakage. The results indicate that storage temperature causes a loss of function in
cell membranes by enhancing lipid peroxidation. A further possibility is to sort the
biological differences of mango fruits to increase the shelf life.
.
211
Summary
212
Samenvatting
Momenteel stijgt de waarde en de productiehoeveelheid van mangovruchten
wereldwijd. Vele studies benadrukken hoe de koudeschade de kwaliteit van
tropische vruchten, zoals mango, beïnvloeden tijdens naoogst behandeling,
vervoer en bewaar. Aangezien de mango één van de meest gewaardeerde en
populaire vruchten in de wereldmarkt en een climacterische vrucht is, d.w.z. snel
rijpend na de oogst, maakt het essentieel te onderzoeken hoe de bewaar de
externe en interne fruitkwaliteit beïnvloedt. Over het algemeen worden lage
temperaturen gebruikt om metabolische processen te vertragen tijdens bewaar om
zodoende het uitstalleven te verlengen. De mango vrucht wordt gekenmerkt door
een korte houdbaarheid bij ongekoelde omstandigheden, maar het is ook gevoelig
voor lage bewaartemperaturen. De gevoeligheid van mangovruchten voor lage
bewaartemperaturen (i.e. onder 12oC) gepaard aan hun bederfelijkheid bij
ongekoelde bewaring beperkt de behandeling, de bewaring en het vervoer van de
vruchten. Om deze redenen moeten de voor- en naoogst factoren die van invloed
zijn op de gevoeligheid van mangovruchten voor kouschade zorgvuldig worden
bestudeerd om het uitstalleven en de kwaliteit van de vruchten te verbeteren.
Het belangrijkste doel van deze studie was de gevolgen te kwantificeren en te
begrijpen van bewaarcondities op het voorkomen van kouschade in
mangovruchten tijdens gekoelde bewaring op lange termijn. Veel aandacht is
besteed aan de integratie van factoren die tot de gevoeligheid van kouschade
zouden kunnen bijdragen en er is geprobeerd om een beter inzicht in de
fysiologische processen te krijgen die met kouschade samenhangen, opdat
kouschade beter begrepen en hopelijk voorspeld kan worden. Het onderzoek
richtte zich op het effect van bewaaromstandigheden (temperatuur en duur van de
bewaring), op mango cultivars (Zibdia en Hindi Be-Sennara), geoogst in de
verschillende stadia van de fruitrijpheid, onrijpe (M1), half-rijp (M2) en volledig rijp
(M3), met betrekking tot de verschijning van kouschade.
Hoofdstuk 1 geeft een overzicht van mango als plant, gewas, en product in
zowel internationale als lokale markten.
Fysiologisch gezien is het effect van lage temperaturen op plantcellen een
fysieke en/of fysiologische verstoring, of verwonding, in celmembranen en het
cytoplasma. Op basis van diverse criteria kunnen deze verwondingen als primaire
dan wel als secundaire verwonding worden gezien. Een primaire verwonding is
een eerste snelle reactie op lage temperatuur die een disfunctie veroorzaakt in de
membranen van de plantencel, in de metabolische processen, of de celorganisatie
Samenvatting
(b.v. cytoskelet). Dit type verwonding kan hersteld worden als de temperatuur
weer boven de schade drempel wordt gebracht. De lange termijn blootstelling aan
lage temperatuur leidt tot de ontwikkeling van secundaire schade of verwondingen
die uit een samenstelling van primaire verwondingen voortvloeien. Bijvoorbeeld als
reactie op het koelen van de cel zullen de membranen faseovergangen vertonen
die in diverse disfuncties zullen resulteren, zoals een verlies van semi-
doorlaatbaarheid, het onjuist functioneren van gebonden eiwitten, het verlies van
membraan gebonden specifieke signaal transductie systemen enz. Op de korte
termijn zijn de gevolgen hiervan omkeerbaar, maar op langere termijn zal de
oxidatie van celcomponenten (b.v. membranen, enzymen) en de metabolische
wanorde zo groot zijn dat het tot celdood door necrose leidt.
Over het algemeen, zal de verschijning van kouschade gepaard gaan met
oxidatieve schade (stress). De oxiderende reacties resulteren in een disfunctie in
de celmembranen door het genereren van AOS (Active Oxygen Species = actieve
zuurstof agentia), oorspronkelijk gevormd door metabole processen. De meest
voorkomende vormen van AOS zijn O2●- ionen en H2O2, die in vivo gevormd
worden door een verscheidenheid aan reakties. Sommige metabole enzymatische
reacties in plantcellen (bijv. de xanthine oxidase en urease) zullen O2●- vormen.
H2O2 kan door dismutatie van twee O2●- ionen worden gevormd, door β-oxidatie
van vetzuren tijdens lipide metabolisme en als gevolg van stress. Een andere bron
van AOS productie is door ziekteverwekkende organismen. Andere delen van de
plantencel kunnen AOS produceren, zoals in verscheidene studies is beschreven,
zoals in het mitochondriele binnenmembraan en in de celwand, maar ook door
enzymatische reacties in de celmembraan. De voortdurende stijging van AOS-
niveaus tijdens bewaring op de lange termijn kunnen ook toegeschreven worden
aan de dalende doeltreffendheid van defensiesystemen, die op enzymatische en
non-enzymatische anti-oxidanten zijn gebaseerd. Non-enzymatische anti-
oxidanten zijn o.a. ascorbinezuur (ASC), α-tocoferol (α-TOC) en β - caroteen (β -
CAR). Deze moleculen zijn vaak verbonden met antioxidant enzymen zoals
ascorbaat-peroxidase, glutathion-reductase, welke antioxidant capaciteit van de
reducerende moleculen regenereren. Zuiver enzymatische reacties, zoals
superoxide dismutase (SOD), katalase (KAT), en de peroxidase (APX) beschermt
de plantcel door O2•− en H2O2 direct af te vangen. De fysiologische veranderingen
worden samengevat in Hoofdstuk 2.
Alle experimenten werden uitgevoerd op twee Egyptische rassen (Zibdia en
Hindi Be-Sennara) die als modellen voor mangofruit werden gebruikt. Drie
verschillende rijpheidstadia op het oogsttijdstip, onrijp, halfrijp en volledig rijpe
vruchten werden gebruikt in dit onderzoek. Er werd 35 dagen lang bewaard bij 5
verschillende temperaturen (2, 4, 7, 10 en 20oC). In alle experimenten werden de
214
Samenvatting
metingen gedaan met tussenpozen van vijf dagen. In een aantal experimenten
werd het effect van het koelen bestudeerd op de water- en lipide-oplosbare anti-
oxidanten en hun enzymen. Diverse kouschade symptomen (zoals
elektrolytlekkage, de hoeveelheid chlorofyl in de schil, de chlorofylfluorescentie
reacties, en een visuele schade beoordeling), en mechanische eigenschappen van
de vruchten werden bestudeerd. Wanneer dat mogelijk was werden zowel de schil
als het vruchtvlees onderzocht. Het doel van deze studies was te onderzoeken
hoe goed de verschillende beoordelingen van kouschade werkten, hoe het koelen
de biochemische eigenschappen van de vruchten veranderde, hoe deze
veranderingen met meetbare indexen van kouschade correleerden, ten einde door
middel van correlatiestudies na te gaan welke anti-oxidanten het belangrijkst
waren voor het ontstaan van kouschade.
Hoofdstuk 3 beschrijft de mogelijkheden om de
Chlorofylfluorescentie parameters te gebruiken (Fv/Fm, F0 en Fm) om de kouschade
van vruchten vast te stellen met het doel de kouschade symptomen te voorspellen
alvorens zij zichtbaar worden. Er is een poging gedaan om het effect van
bewaartemperatuur op verschillende mango rassen in verschillende rijpheidstadia
te analyseren. De vruchten werden 35 dagen bewaard bij 5 temperaturen (2, 4, 7,
10 en 20oC). De kouschade index (CI-index) en de concentratie chlorofyl a, b en
de chlorofyl a/b verhouding werden elke 5 dagen gemeten en de
chlorofylfluorescentie (CF) om de 3 dagen. Er werd aangetoond dat de
temperatuur en de rassen met de bewaartijd een significant effect hebben, maar er
werd geen effect van rijpheidstadium waargenomen. Een hogere
bewaartemperatuur verminderde Fv/Fm en Fm, terwijl F0 tijdens het experiment
steeg. Tijdens een verlengde bewaring bij lage temperatuur verminderde de Fv/Fm
waarde sneller, aan het eind van het experiment, in Zibdia dan in Hindi vruchten.
In Zibdia nam het na 3 dagen af en in Hindi na 6 dagen en dit alles vóór de
zichtbare symptomen van kouschade konden worden waargenomen. In alle
rijpheidstadia liet Zibdia zwaardere kouschade symptomen tijdens bewaring zien
dan Hindi. Kouschade verscheen op dag 10 in beide rassen in alle rijpheidstadia.
Het was minder zwaar in M1 vruchten dan in M2 en M3. Chla brak sneller af in
Zibdia dan in Hindi en sneller dan Chlb in alle rijpheidstadia. Er verdween meer
chlorofyl in Zibdia dan Hindi. Deze verhouding staat een voorspelling van
kouschade toe voordat de zichtbare symptomen verschijnen. De resultaten tonen
aan dat de Zibdia vruchten gevoeliger zijn voor lage bewaartemperaturen dan
Hindi vruchten. Daarom is het noodzakelijk te onderzoeken welke factoren de
weerstand tegen lage temperaturen verhoogt.
Het effect van bewaartemperaturen op water- en lipide-oplosbare anti-
oxidanten, zoals ascorbinezuur (ASC), α-tocoferol (α-TOC) en β-caroteen (β-CAR)
215
Samenvatting
concentraties tijdens bewaring en de correlatie met CI-Index wordt beschreven in

Hoofdstuk 4. Het experiment werd uitgevoerd op twee mango rassen `Zibdia' en
`Hindi', welke in drie verschillende rijpheidstadia werden geoogst. De vruchten
werden opgeslagen bij verschillende lage bewaartemperaturen, met het doel een
beter inzicht in de onderliggende fysiologische processen van de ontwikkeling van
kouschade met betrekking tot anti-oxidanten die een bescherming tegen
schadelijke actieve oxiderende reagentia (AOS) te krijgen. Vanuit fysiologisch
perspectief verhoogt de langdurige bewaring de uit evenwicht situatie tussen
oxiderende en de reducerende reagentia, welke tot verhoogde oxidatieve schade
aan celmembranen, na reacties met membraan lipiden en eiwitten, leidt. Dit
resulteert definitief in celdood. Daarom werden gedurende de bewaarperiode
water- en het lipide-oplosbare anti-oxidanten gemeten in vruchtdelen (schil en
vruchtvlees). Over het algemeen werd van beide rassen een hogere begin ASC
concentratie in M1 vruchten waargenomen in vergelijking met de M2 en M3
vruchten. α-TOC en β-CAR toonden de tegenovergestelde tendens. Het laagste
niveau van ASC werd waargenomen in bijna compleet beschadigde vruchten. Een
snelle dalingen van ASC, werd waargenomen in de half (M2) en volledig rijp (M3)
fruit en bij de vruchten die bij de lage temperaturen werden opgeslagen. Een
negatieve lineaire verhouding werd gevonden tussen water/lipide-oplosbare anti-
oxidanten en de CI-Index hetgeen bewijst dat water/ lipide-oplosbare anti-
oxidanten af nemen op basis van de bewaartemperatuur, de bewaarduur en het
rijpheidstadium.
De gegevens die in het vorige hoofdstuk werden verkregen staan ons toe
om de activiteiten van anti-oxidant enzymen te bestuderen (SOD, CAT, APX en
GR) en de eindproducten van AOS oxydatie van lipiden en eiwitten die tijdens
bewaring voorkomen. In dit experiment werden Zibdia en Hindi 35 dagen
opgeslagen bij twee bewaartemperaturen (4 en 10oC). De resultaten wijzen erop
dat de lipide-peroxidatie en de eiwit-oxidatie in beide rassen en in alle vruchtdelen
tot aan het eind van het experiment toenemen. De activiteit van de anti-oxidant
enzymen steeg tot het maximum, afhankelijk van de bewaartemperatuur, de
rassen, het rijpheids stadium en de bewaarduur. Bovendien werden in de
verschillende vruchtdelen verschillende enzymactiviteiten voor SOD, CAT, APX en
GR, in relatie tot de bewaartemperatuur. Er werd een negatief lineair verband
tussen de MDA concentratie en de activiteit van anti-oxidant enzymen (KAT en
APX) waargenomen. Dit verband toont de onevenwichtigheid tussen anti-oxidant
activiteit en de AOS vorming, welke door stijgende lipideperoxidatie in zowel
mango rassen als vruchtdelen wordt weerspiegeld. Analoog werd een negatief
lineair verband tussen eiwitoxidatie (PCG) en anti-oxidant enzymactiviteiten (KAT,
APX en SOD) gevonden.
216
Samenvatting
Hoofdstuk 5 beschrijft de ontwikkeling van fysiologische veranderingen

zoals ethyleenproductie (C2H4), ademhalingssnelheid (CO2), stevigheid en
ionenlekkage in mango cultivars. De resultaten wezen erop dat de climacterische
piek in de ademhaling op dag 10 van de bewaring werd waargenomen. Bovendien
bereikte de ethyleen productie een maximum op dag 5 van bewaring. M3 vruchten
genereerden de hoogste hoeveelheden C2H4 in vergelijking met M1 en M2
vruchten in beide rassen. Hetzelfde patroon werd waargenomen t.a.v. de
ademhalingssnelheid. De bewaartemperatuur beïnvloedt de C2H4 productie en de
ademhalingssnelheid (CO2), welke hoger zijn bij 20oC dan bij lage temperaturen (2
aan 10oC). Bewaring bij lagere temperaturen behield de vrucht stevigheid beter bij
beide rassen. Hindi vruchten waren steviger dan Zibdia vruchten en M1 vruchten
waren steviger dan M2 en M3. Een ander effect van de bewaartemperatuur dat in
dit experiment werd waargenomen is dat de ionenlekkage bij lagere
bewaartemperaturen toenam, dit duidt op het disfunctioneren van de membranen
en de cel wanden in de plantcel. Er werd een verband waargenomen tussen de
producten van de lipide peroxidatie (MDA) en de ethyleenproductie,
ademhalingssnelheid, de stevigheid en de ionenlekkage. Deze verhoudingen
wijzen op het disfunctioneren van het celmembraan, wat toe te schrijven is aan
verhoogde peroxidatie processen in het membraan. MDA had een negatieve
relatie met ethyleen (C2H4) productie en de vruchtstevigheid. Uit deze
verhoudingen is af te leiden dat de bewaartemperatuur een belangrijke rol vervult
in de toename in lipide peroxidatie processen en het remmen van de hydrolytische
enzymen, welke in verband staan met het zacht worden van de vrucht. Hoewel
anderzijds een negatieve relatie werd gevonden tussen MDA en de
ademhalingssnelheid (CO2) en de ionenlekkage. De resultaten wijzen erop dat de
bewaartemperatuur een verlies van functionaliteit in celmembranen veroorzaakt
door lipide peroxidatie processen te intensiveren. Een andere mogelijkheid is het
sorteren van de biologische verschillen van mangovruchten om het uitstalleven te
verhogen.
217
Samenvatting
218
‫اــ اــــ‬
‫ا‪:‬‬
‫ا ا ا ا ا وذات * ا* )د '& ا‪%‬ق ا ! و ا"! أ‬
‫‪123 %4‬ض ‪ 4‬ارة ا ‪ ' 5"6 72‬ات "! وأن ‪72‬‬ ‫و* أ‪.‬رت ارات ان ا‬
‫‪9‬ر ا ‪ 5"6‬درا‪:‬ت ‪ 4‬ارة ا* ‪١٢‬م ا‪@? 5‬ر ?ه ة ا‪ 6‬اض )‪ (Chilling injury‬ا ودة‬
‫‪ 5"6‬ا‪J‬ر ‪I‬دى ا‪"6 5‬ت ا اول و ا او ا‪ F‬وآ‪ "6 CD‬ا ‪ .%‬آ ث ه‪D‬ة‬
‫ا‪O‬ه ة '‪ 5‬ا‪ 1" 2 4‬ا‪ 4‬ا ‪N‬ت ‪ M‬ا)د ‪ 5"6 9I‬اد‪ Q‬ا ‪:%' .P‬‬
‫‪I‬دى ا ‪ 5"6 72‬درا‪:‬ت ‪ 12‬ا‪S7: 5‬ت اآ‪ .(radicals Free) RF %‬ا ‪ 5‬‬
‫‪ U" 2 V‬ا آ ت ا دا‪ T‬ا‪ "2‬ا ‪ .‬وآ ه‪D‬ة ا‪S7‬ت '‪ 5‬آ‪ N‬ا‪ "2‬ا و‬
‫اا‪ .‬ه‪D‬ة ا‪S7‬ت ‪ 1" 2‬دا‪ T‬ا‪ "2‬ا ‪ 6‬ا ‪N61‬ت ا‪ 73‬و‬
‫‪ W‬ا‪ 73‬ا‪ 9I‬ات ا‪2‬ر‪ 5"6 :‬ا‪ ."2‬ا‪ 9I‬ات ا ‪ 5"6 6% 5‬ه‪D‬ة ا‪S7‬ت ‪J‬‬
‫ا"‪ ,4‬ا‪1‬ف‪ ,‬أ‪12‬ض او ار ‪1‬ع در‪ :‬ا ارة‪ .‬اآ ا ‪ @' 5‬ه‪ 5‬ار ا‪,"2‬‬
‫ا‪ FW‬ا ‪N‬ز‪ ,‬ا آر‪ ,‬ا]"رو‪ ,\NM‬ا‪NM %‬زم‪ .‬و‪)2M‬ص `ع ا _ ا‪ R‬وح ه‬
‫أن ‪ 72‬ا‪ 5"6b‬در‪:‬ت ‪ 4‬ارة ‪ 12‬ود " ‪ 5"6 6%‬زدة ‪Free radicals‬‬
‫و‪ V‬ا ار ا ‪I 72‬دى ا‪ 5‬زدة ‪ %‬ى ه‪D‬ة ‪ Free radicals‬دا‪ T‬ا‪ "2‬اذ ‪ V 61‬ا و ‬
‫و اهن ا‪ FW‬ا ‪N‬ز ‪ @)))T 1‬وو?‪ 5' @1‬ا‪ 4‬ا ‪ .72‬و‪:‬‬
‫ا‪].‬ل ‪ 1" 2‬ا‪S7‬ت اوآ‪ 61 (NO,H2O2, O2—, and HO) :J %%‬ا‪S7‬ت‬
‫‪ V‬اه‪ c‬آ ت ا‪ %W‬ا ‪N‬ز ا و و اهن وا] @ وا ‪"%" 5' (DNA) V‬‬
‫ ا ‪N61‬ت‪ .‬ا ‪ P‬ه‪D‬ة ا ‪N61‬ت ] ه و*@ آ‪ .I‬ات دا ‪: 5"6‬وث ف‬
‫ا@ف اول '‪N61 5‬ت اآ‪%‬ة ا ‪dF 5‬‬ ‫‪NW1 M‬ت اآ‪%‬ة )‪ .(Oxidative Stress‬اهن‬
‫‪NT‬ل ‪ 72‬ا‪J‬ر‪ .‬ار ا ‪ 5"6 72‬در‪ 4 :‬ارة ‪ % 12‬ه‪D‬ة ا ‪N61‬ت ‪I‬دى‬
‫ا‪ 5‬ت ا‪ ,"2‬و‪ @O 5 M‬ا‪ 6‬اض‪ C" U* .‬ا ‪N61‬ت اآ‪%‬ة‪7" _4‬م ا‪ "2‬ا‪ 5‬و‪:‬د‬
‫ات ااآ‪%‬ة )‪ (Antioxidants‬و@ ا آ ت أ‪J 7‬ل ) ‪antioxidant enzymes:‬‬
‫‪ (SOD, CAT, APX and GR‬و‪ W‬أ‪non-enzymatic: Ascorbic acid, α-) &7‬‬
‫‪ (Tocopherol and β-Carotene‬اذ ‪7 " Free radicals V 61‬ا ‪ %‬اه دا‪T‬‬
‫ا‪ ."2‬و‪:‬د ا ازن ‪ % M‬ى ‪ Free radicals‬و آ‪ 5 antioxidants 7‬ا‪4 "2‬‬
‫وأ‪N T‬ل ه‪D‬ا ا ازن ‪3‬ى ‪ 4 J 9I‬ارة ا ‪ 72‬أ ‪N61‬ت ا‪3‬آ‪%‬ة ‪Oxidative Stress‬‬
‫و?@ر أ‪ 6‬اض ا ودة‪.‬‬
‫@ف ارا ه ا‪ 5‬ا " ا]‪ 5‬و ‪ ? 9b c@1‬وف ا ‪4 5"6 72‬وث ?ه ة ) ‪Chilling‬‬
‫‪NT (injury‬ل ا ‪ 1 72‬ات "! ‪ 5"6‬درا‪:‬ت ‪ 4‬ار‪ !12 Q‬أ* ‪o12‬م‪ .‬آ @ف ارا‬
‫ا‪ 5‬ا‪3‬ه م ‪ M‬ا ا ‪] * 5‬ن @ ‪@OM !*N6‬ر و ‪R‬ر ه‪ QD‬ا‪O‬ه ‪ .Q‬و ‪ c@1‬ه‪ QD‬ا‪O‬ه ة ‪g"R‬‬
‫ا‪ 5‬ا ‪N 1‬ت ا ا ‪ 5‬ث ‪NT‬ل ا ‪ 72‬وا ‪I 5‬دى ا‪@? 5‬ر أ‪ 6‬اض ا‪ M‬وذ‪b " C‬‬
‫‪M‬وث ه‪ QD‬ا‪O‬ه ة‪ .‬وآن ا آ‪ 5"6 7‬ا ا ا ‪ @ 5‬دور آ ه‪ 5‬در‪ 4 :‬ارة و*\ ا ‪5"6 72‬‬
‫‪9‬ر ا‪.‬‬
‫ورا ه‪D‬ة ا‪O‬ه ة ‪ g"R‬ا‪ 5‬أ‪ :‬اء ‪ 5"6 _M‬ا‪ 4‬ا‪1‬آ@ ا ا‪ 3‬ا ‪ J‬ا‪b‬‬
‫وه‪` 5‬ع ارا وا و' ‪bM‬ه @ ا‪) *3‬د ‪ 5"6‬ا‪ %‬ى ا‪%‬ق او‪ 5‬و ا ‪ 5M‬و‬
‫ا) ى‪ .‬وا‪ Q 3‬ه أ‪ :‬اء ‪ i M‬ا رب " ‪ i M c‬ا‪3‬ف ا‪ b‬ا) ‪J‬ل ا‪ MD‬و‬
‫ا@ى ‪%M‬رة ' ى او! او ا‪ 5"6 72 " %‬در‪:‬ت ‪ 12‬و آ‪ CD‬ا ‪M I‬وث‬
‫‪@? * Chilling injury‬ر ا‪ 63‬اض ا‪ .M3‬وا '! ‪ %‬ى )‪Ascorbic acid, α-‬‬
‫‪ (Tocopherol and β-Carotene‬وآ‪F CD‬ط ) ‪antioxidant enzymes: SOD, CAT,‬‬
‫‪ .(APX and GR‬وآ‪* CD‬س ا ‪N61 P‬ت ا‪3‬آ‪%‬ة ‪ .Oxidative Stress‬و \ ا رب ‪5"6‬‬
‫ا‪ b‬ا ‪)4 c 5‬ده ‪N9 5"6‬ث أ‪6‬ر ‪(Immature, Half mature and Full !1" 2‬‬
‫)‪ mature fruits‬و ‪ mT 5"6 @72 c‬در‪:‬ت ‪ 4‬ارة )‪°20 ,10 ,7 ,4 ,2‬م( ‪ 1‬ة ‪ 35‬م ‪V‬‬
‫آ ات ا‪ 1" 2‬ا‪ M"R‬آ ‪ m4‬أم ا‪ 1‬ة ا]" " ‪ 72‬و'‪ 5‬أ‪7:‬اء ‪ 1" 2‬ا‪ J‬ة‬
‫)ا‪ F‬ة و ا"‪.(g‬‬
‫او‪ :3‬أ ‪2‬ام *س ‪F‬ط ا]"ر' )‪Chlorophyll Fluorescence (CF‬‬
‫ا ‪2‬ام ه‪D‬ة ا ‪@M‬ف ‪ c‬ى ا‪ 72 " %‬وآ‪ CD‬ا ‪M I‬وث ?ه ة ‪Chilling injury‬‬
‫* ?@ر ا‪ 63‬اض ا‪ M3‬آ ‪ c‬و‪ 5' 1‬ا‪7‬ء ا‪ ._J‬أذ أ! ‪ c‬ى ا‪ %‬أف‬
‫ا‪123 b‬ض در‪ 4 :‬ارة ا ‪ \ .72‬ه‪D‬ة ارا ‪ 1‬ا‪) b‬ا‪7‬رو‪\ 6‬‬
‫ا‪ O‬وف ا) (‪ UR* c .‬ا‪J‬ر ا)‪) 1‬ا‪ MD‬و ا@ى ‪%M‬رة( ‪N9 5"6‬ث أ‪6‬ر‪ .1" 2‬ور‪56‬‬
‫‪ i M‬ا‪ :‬اءات ا‪9‬ء ا‪) UR‬ا‪ T‬ر ا‪.‬ر و ا‪J‬ر( آ‪ CD‬ا‪ 4‬ت ا‪ 5' M"R‬ا‪J‬ر‬
‫و‪ @.‬ا‪ 5‬ا"] ا@‪ .‬و وا*‪ V‬ا ‪ P‬ا ) ‪ @"6‬أن ‪ u4‬أن ‪F‬ظ ‪ CF‬أ‪FM i12‬ة‬
‫‪ 3 M‬أم '‪ 5‬ا‪ MD‬و ‪ 9‬أم ا@ى و ‪ °2 5"6 @72 6 )T‬م و‪°4‬م ‪N2M‬ف ا ‪5"6 72‬‬
‫‪ °7‬م و‪°10‬م ا ا ‪° 20 5"6 72‬م آ‪b‬ن ا‪123‬ض ‪.‬ا‪.‬آ ‪ u4‬أن ‪ U‬ا‪ " b‬ا‪"MD‬‬
‫‪%4‬س " ‪ 5"6 72‬در‪ 4 :‬ارة ‪N2M 12‬ف " ا@ى" وذ‪ C‬وا`‪ x‬ات ا‪Tb‬ذة ‪NT‬ل‬
‫' ة ا ‪ .72‬وأ ‪ u4‬أن ا‪ 6‬اض ا‪ Chilling injury M‬أ ‪ 6‬ام ا ‪ .‬ا ‪.72‬‬
‫‪R‬ر ه‪D‬ة ا‪ 6‬اض ‪7M‬دة ' ة ا ‪ .72‬و‪ dM M‬ا ‪ P‬ا ) ‪ @"6‬أن آ ‪ CF‬و ‪Chilling‬‬
‫‪ x injury index‬أن ‪ U‬ا‪ . M7‬ا‪ 5"6 72 " %‬در‪:‬ت ا ارة ا‪6 12‬‬
‫ا@ى‪ .‬آ ‪. U" 2‬ة ا‪ 6bM M3‬اض ا ودة‪ g%4 5"6‬ا)‪ U‬وآ‪ CD‬ا ا‪J‬ر‪ _4 .‬آ‪\b‬‬
‫ا‪. M‬ة '‪ 5‬ا ا‪ 6 (Full mature) _J‬ا ا‪ (Half mature) 5bJ‬و اول‬
‫)‪ 5' (Immature‬آ‪ N‬ا)‪ 1‬و] ا‪. M‬ة '‪ 5‬ا‪ 6 MD‬ا@ى‪ .‬و وا*‪ V‬ا ‪ P‬ه‪D‬ة ]‬
‫ال ‪bM‬ن ‪ U‬ا‪%4 MD‬س اآ‪ U J‬ا@ى‪ .‬واذ ] ا ‪M I‬وث ا‪NT M3‬ل‬
‫ا‪123‬ض ا‪@? * CF 5' F‬ر ا‪ 6‬اض ا ‪ 5"6‬ا‪J‬ر‪ .‬و‪` 5"6‬ء ه‪D‬ة ا ‪bM P‬ن‬
‫ا‪'NT‬ت ‪ M‬اف و ا‪63‬ر ‪ g"R‬ا‪ 5‬أ‪ :‬اء رب ا‪" 6‬را ا‪ 1‬ق ا‪NT 5:%1‬ل‬
‫درا ‪ %‬ى ‪.Antioxidants‬‬
‫‪ :ًb9‬دات ا‪3‬آ‪%‬ة ا‪ z‬أ‪Non-enzymatic antioxidants 7‬‬
‫درا ‪ %‬ى دات ا‪3‬آ‪%‬ة '‪ 5‬آ ا‪ MD‬و ا@ى @ف ا‪ % *N6 c@' 5‬اه و?@ر و‬
‫‪R‬ر أ‪ 6‬اض ا ودة ‪N4‬ل ' ة ا ‪ 35) 72‬م(‪ .‬ه‪D‬ة ادات اآ‪%‬ة ‪Ascorbic acid‬‬
‫ا وف ‪M‬ـ‪ C 1‬و ‪ (E ') α-Tocopherol‬و ‪ c@ (A ') β-Carotene‬‬
‫دور آ '‪ % i1T 5‬ى ا ‪N61‬ت اآ‪%‬ة ا‪9‬ء ا ‪ \ .72‬ه‪D‬ة ارا ‪ 5"6‬آ ا)‪1‬‬
‫ا‪` b‬ع ارا و‪ 5"6‬أ‪6‬ر ‪ 72 c .1" 2‬ا‪J‬ر ‪ mT 5"6‬درا‪:‬ت ‪ 4‬ارة )‪,7 ,4 ,2‬‬
‫‪°20 ,10‬م( ‪ 1‬ة ‪ 35‬م‪* c .‬س ‪ %‬ى ‪ Antioxidants‬آ ‪ mT‬أم ‪ 1‬ة ا ‪ 72‬آ ‪c‬‬
‫‪218‬‬
‫ة ادات‬D‫ ى ه‬V‫ ز‬R T C‫( وذ‬g"‫ ة و ا‬F‫ ة )ا‬J‫ ا‬1" 2 ‫اء‬7:‫ ا‬5' @*
.‫ر أ‬63‫ اف وا‬M ‫ اض ا ودة‬6‫ر أ‬R ‫* @ ى‬N6‫و‬
‫ ى‬5' ‫ و ا@ى‬MD‫ ا‬M ‫ف‬N T‫ أ‬: !‫ أ‬M ‫ة ا‬D‫"@ ه‬6 ) ‫ ا‬P ‫ل ا‬
‫ ان‬u4 ‫أذ‬.4‫ اا‬U)‫ر ا‬6‫ أ‬M ‫ف‬N T‫ ا‬u4 CD‫ `ع ا _ آ‬9NJ‫ ا‬Antioxidants
‫ ا اول و‬56 Ascorbic acid ‫( اه‬immature fruits) 5‫ر او‬6‫ ى ا‬
‫ أذ‬β-Carotene ‫ و‬α-Tocopherol ‫ف ى‬N2M (Half and full mature fruits) 5J‫ا‬
5"6 ‫ ا م‬5' MD‫ أن ا‬u4 ‫ آ‬._J‫ ا‬5' 5"6‫ وا‬5J‫ ا ا‬5' ‫داد‬7‫ ا اول و‬5' *‫]ن أ‬
‫ ا‬β-Carotene ‫ ى‬% CD‫روآ‬6‫ ا‬V: 5'‫ ا@ى و‬6 Ascorbic acid 5"6‫ ى أ‬
5‫ ا‬%M
‫ة ادات‬D‫ ى ه‬% ‫ أن‬u4N‫ و ا‬.MD‫ ا‬6 ‫ر ا@ى‬9 5' 5"6‫ ' ى أ‬α-Tocopherol
‫ ا‬5' *‫ ا‬M‫ اض ا‬6‫ أن ?@ر ا‬P ‫ ا‬V*‫ وا‬u4 ‫ وا‬.‫ر‬J‫ ا‬72 ‫دة ' ة ا‬7M
*‫ ا‬7‫ و آ‬Ascorbic acid 5"6‫ ا‬7‫ آ‬5"6 ‫ أن ا@ ى‬V ‫ا‬D‫ ى وه‬T‫ر ا‬63‫ ا‬6 ‫اول‬

‫ ه و‬u4N‫ ا‬.1)‫ ا‬N‫ آ‬5' _J‫ و ا‬5J‫ ا ا‬m] M ,β-Carotene‫ و‬α-Tocopherol
‫ر ا اول‬9 ‫ ذا‬:‫ر‬6‫ ا‬M CD‫ اف وآ‬M :M ‫'ت‬N T‫ ا‬: !‫ء ا‬7‫ا ا‬D‫ل ه‬NT
V:‫ا را‬D‫_(؟ وه ه‬J‫ و ا‬5J‫ر ا* )د )ا‬6‫ ا‬6 (immature fruits) *‫ ا‬M‫ا?@ ت ا‬
‫ة‬D‫ ه‬.‫ر‬6‫ ا‬M (β-Carotene ‫ و‬α-Tocopherol Ascorbic acid) ‫ف ا ى‬N T‫ ا‬5‫ا‬
‫ او‬Chilling injury ‫ــ‬M M‫ة ا‬. " 5' @‫ا‬2 ‫ ا] ا‬6 ‫ ات‬.I @ ‫ف * ]ن‬N T‫ا‬
.M‫ون اى ا‬M ‫ر‬J" ] ‫ ال ة‬5‫ ا‬72 ‫ذدة ' ة ا‬
Antioxidantsِ enzymes 7‫ة أ‬%‫آ‬3‫ دات ا‬:ًJb9
‫ة‬%‫ط دات اآ‬F ‫ درا‬5‫ ا‬Q ‫ ا‬5‫\ ا‬7‫ ا‬M%‫"@ ا رب ا‬6 ) ‫ ا‬P ‫ ا‬
‫ ?@ر‬M ‫ت * و‬7‫ة ا‬D‫ط ه‬F c@1 ‫@ف‬M (SOD, CAT, APX and GR) J 7‫ا‬
M ‫ة ا‬D‫ ا م ه‬.‫ر‬J" "]‫ ا‬72 ‫ل ' ة ا‬NT ‫ ى‬M ‫ط‬F‫ ا‬C‫ ذ‬dM‫ و ا] ر‬M3‫ا‬
:‫ در‬5"6 72 ‫ة وا‬. M‫ ا‬g % ‫م‬°4 ‫ ارة‬4 :‫ در‬5"6 72 ‫ن ا‬bM M%‫" ا رب ا‬R
5' 7‫ط دات ا‬F ‫ *س‬5' ‫ ']ن اه م‬F‫"ت ا و ا‬6 5' J‫ ا‬5‫م ه‬°10 ‫ ارة‬4
(g"‫ ة وا‬F‫ر)ا‬J‫ت ا‬6 DT‫ أ‬V ‫ م‬35 ‫ر ة‬J‫\ ا‬7T .1" 2 ‫ر‬6‫ أ‬5"6‫ و‬1)‫ ا‬m1
‫ ا ازن‬5' "T 6 ‫د‬:‫ و‬5‫ ا?@ ت ا‬%] ‫*ت ا‬N ‫ آ أن ا‬.‫ت‬7‫ط ا‬F ‫ أم س‬mT ‫آ‬
5' !`‫دة اا‬7‫ ا‬x`‫ا وا‬D‫ وه‬AOS ‫ و‬antioxidant enzymes activity‫ ى‬% M
‫ر‬J‫اء ا‬7:‫ ا‬CD‫ و ا@ى وآ‬D‫ ا‬1)‫ آ ا‬5' 72 ‫ل ا‬NT lipid peroxidation
antioxidant enzymes activity‫ و‬PCG M ‫\ ا‬O4 Q ‫ ا‬m1 V ‫( و‬c"‫ ة و ا‬F‫)ا‬
.(CAT, APX and SOD)
Ethylene, respiration and firmness and ion leakage MN)‫ وا‬m1 ‫" وا‬9‫ ا‬:b M‫را‬
‫ و‬m1 ‫ و ل ا‬C2H4 ‫ ا ج‬J :%1‫رات ا‬R ‫ ا ول ا‬m2‫ء ا‬7‫ ا‬5'
‫ ام‬5' m1 ‫ ان ذروة ا‬u4 Climacteric respiration ‫ أن‬P ‫ ا‬F ‫ و‬.!‫ذ‬1‫ و ا‬MN)‫ا‬
‫ ت آ‬U" 2‫ و‬.M ‫ ا‬m2‫ ام ا‬5' "9‫ ل ا ج ا‬5"6‫ ا‬CD‫ آ‬M ‫ ا‬. ‫ا‬
219
‫ا‪ "9‬و ا ‪ g%4 5"6 m1‬ا‪6‬ر ا‪J‬ر ‪] _4‬ن أ* '‪ 5‬ا اول و ‪D‬داد ‪ V‬ذدة ا ‬
‫ا‪ J‬ة‪ .‬آ ‪ u4‬ان ا ‪ 5"6 72‬درا‪:‬ت ا ارة ا‪ MN 5"6 u' 12‬ا‪J‬ر '‪ 5‬آ"‪5‬‬
‫ا)‪ .1‬أ ‪ u4‬ا)‪ MN‬أ‪ 5' 5"6‬ا اول و ذدة ا ا‪ J‬ة‪.‬‬
‫ذدة ‪ ion leakage‬ا‪12‬ض در‪ :‬ا ارة ا ‪ F 72‬ا‪4 5‬وث ‪ 5' "T‬ا‪FW‬‬
‫ا ‪N‬ز و ار ا‪ "2‬أ‪9‬ء ا ‪ .72‬و ‪ \ J‬ا ‪*N‬ت ‪ lipid peroxidation M‬و ا‪ "9‬و‬
‫ا ‪ m1‬و ا)‪ MN‬و ‪ F _4 ion leakage‬ا ‪*N‬ت ا‪ 5‬ا‪ 5' "2‬ا‪ FW‬و ار ‪ V:‬ا‪ 5‬ذدة‬
‫‪ lipid peroxidation‬ل ‪ 5"6‬در‪ 4 :‬ارة ا ‪ g " 72‬دور '‪ 5‬ذدة ‪lipid peroxidation‬‬
‫و ‪ d J‬ا‪7‬ت ا " ) ‪ (hydrolytic enzymes‬وا ‪ @ 5‬دور '‪ 5‬ا‪J‬ر‪ .‬و ا ‪ *N‬ا ]‪!%‬‬
‫ا ‪ 5‬و‪:‬ت ‪ lipid peroxidation M‬و ا ‪ F m1‬ا‪ ' 5‬ا?‪ U‬ا‪ FW‬ا‪ 6 !"2‬‬
‫" اهن‪ .‬أذ ] ا ‪1‬دة ه‪D‬ة ا‪'N T‬ت ا ‪J :‬ر ا ‪7‬دة ا ا ‪.72‬‬
‫‪220‬‬
Acknowledgment
Firstly, I would like to thank ‘Allah’ who gives me a power and success to do this
work for science. I had got my scholarship to study my PhD abroad I had two options to
go UK or USA since 1996. In this year I met Dr. Anton De Jager in my department in
Mansoura University. I hared from my college Dr. Awad many good things about
Netherlands. Also from Dr. Dixon from Selso Collage, Bradford he convinced me to go.
Even my farmers in my village said to me very good issues about Holland. From this
moment, I decided to change my direction to come to Netherlands instead of UK and
USA. Thank you Dr Anton a lot, for opening the door for me to come to Wageningen
University.
I would like to thank my promoter Professor Olaf van Kooten for his supervisory,
guidance me from the moment to me in The Netherlands also my appreciation to my
daily supervisor Dr. Jeremy Harbinson for his supervisory, gentleness and guidance me
in my work also his helping for solving problems during my lab experiments also for his
efforts for writing my thesis, Thank you Jeremy for that. I would like to exploit this
opportunity to express my confidential grateful to group of HPC Department of
Horticultural Production Chain Wageningen University, the Netherlands, for providing a
congenial condition to work during my five years. I am appreciating to a good advice I
had got from Dr. Ep Heuvelink for doing statistical analysis, he always gives me time to
discuses and his reading chapter 4, also Dr Uulke van Meeteren on reading chapter 1
and 3, Dr Wim van Ieperen for reading chapter 6 and Arjen van de Peppel, thank you, I
learned a lot. I would like to appreciate Prof. Dr. Luinus van der Plus, in Plant physiology
Wageningen University for supporting me and Dr. Jan Rijstenbil, from Department of
Marine Microbiolog, Netherlands Institute of Ecology, for his guidance and help as for
chapter 5. Meanwhile, I would like to thank all my PH.D. Colleges (Dr. Megual costa,
Lee and Susana) for there advices to me and my family and the colleague Dr. Milza
Lana for here joining me work, fun and walking around. I am grateful to acknowledge my
“brother, friend and colleague’’ Hossein Rezaeinejad for his standing beside me all time.
Also, my appreciate to my paranimphs Peter Twumasi and Hossein Rezaeinejad for
their help.
I would like also to express my appreciates to Egyptian embassy members in
Den haag, Netherlands, Ambassador, El-habashi, N and Consulate Walid Aldin for
there support during last two years also their official arrangement in Netherlands and
Egypt. Also I am appreciate to all efforts by Egyptian embassy in Germany, especially
223
Prof. Alia Khatab for here help and arrangement in High educational ministry and my
University until to finalize my work.
I am appreciating to the academic stuff of the Pomology Department of Faculty
Agriculture, Mansoura University for supporting me all time to get a scholarship, from the
Egyptian of High Educational Ministry, also during my staying in Netherlands until finish
my work. Especially, Prof. Iraqi. M; Prof. El-boray, M.S.; Prof. Fahmi M.; Prof. Hegazi,
A.; Elkady M. Samra N.; Elbana G.; Saman L. G.; Dr. Mohamed Awad. I am also
grateful to thank Prof. Elwakel, M; Head of pathology department Facility of Agriculture,
Mansoura University for valuable discussion and his supporting during my staying.
I would like also to thank my Egyptian friends; I met here in Wageningen
University Dr. Hamed Emashed (Mansoura Univ.); Dr. Tamer Gamel (Alexandria Univ.);
Salah Elasal (Cairo Univ.) and Tarik Rabie (Suse Chanal Univ.). Also some Arab
friends, Mr. Moyyed M; Mr. Akram, B.; Mr Adale and Mr Nabil for giving me help in
Wageningen.
My great appreciation and deep gratitude are due to my father for supporting me
during my staying in The Netherlands. Also my college Dr. Refay Eldengawy in my
department in Mansoura university for supporting and helping me to complete my
experiments; also, I would like to thank Eg. Ahmed Abd El-manam for his supervisory for
importing my fruits materials to Netherlands in Cairo airport.
Last, I would like to express my deeply thanks to my parents, relatives, mother
and father in low for their emotional support. I would like also to express my thanks and
appreciations to my wife; even it is not enough for here to express here right who join
me all difficulties and happiness moments during here staying with me in Wageningen.
Finally, to the best events in my life is my little wild flowers Lojina and Norssen.
_Étç TÜtytà
224
Curriculum vitae
Loay Arafat was born in Mansoura city, Egypt on 10th November 1969. After he
fished high school in 1989, he started studying Agriculture (Plant production) in,
faulty of Agriculture, Mansoura University, Egypt. The B.Sc. qualification was
obtained in 1992 with majors in horticulture, agronomy, and pathology and plant
protection. After that, he asked by Horticulture Department of faculty to work as a
teaching assistance, he took the responsibilities of doing research and teaching
within this department. In the same period he started his M.Sc. program in
pomology within the Horticulture Department in fruit production under supervision
Prof. Iraqi, M; Prof. El-Boray, M. and Dr. Fahmi M. He did an experimental research
for M.Sc. degree in Pomology Department, Mansoura University. It is titled ‘a
comparison between foliar and soil potassium fertilization on Thompson seedless
grape. After that, he employed by the same department as an assistance lecturer
(Mansoura University in June 1996).
In 1996, he got a scholarship from High Educational Ministry, Cairo to study in field
postharvest fruit quality at Wageningen University and Research Centre in
Horticulture Production Chains Group, The Netherlands. In 1999, he started his
Ph.D. work. He was also contributed to many international symposium and
workshops
Currently since 2005, Loay Arafat has a position at the Pomology Department
Faculty of Agriculture Mansoura University, Egypt.
His address in Egypt is:

Loay Arafat
Pomology Department Faculty of Agriculture
Mansoura University
El-Mansoura/ Egypt
Box 35516
Phone:+20-50-2268606 – 2236002 –2245274

Fax :+20-50-2221688 – 2245268
E-mail: loayArafat@mans.edu.eg
Home page: http://www.mans.edu.eg/FacAgr/english/
225
226
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Chilling injury in mangoes

Article in Wageningen Agricultural University Papers · October 2005

The user has requested enhancement of the downloaded file.

Loay Abd Ellatif Taha Arafat

Prof. Dr H.J. Wichers (A&F)

Prof. Dr Ir P.C. Struik (Wageningen Universiteit)

Dit onderzoek is uitgevoerd binnen de onderzoekschool Production Ecology &

Loay Abd Ellatif Taha Arafat

ter verkrijging van de graad van doctor

Horticultural Production Chains Group, Department of Plant Sciences, Wageningen

Chapter 3 Chlorophyll fluorescence assessment of chilling injury in ‘Zibdia’ 53

Chapter 4 Changes in the concentration of water and lipid-soluble 83

Chapter 5 Antioxidant enzymes and oxidative stress in mangoes 119

Chapter 7 Final comment 191

Botanical and taxonomical of mango

Figure 2. Tree Figure 3. Different leaves maturity

Figure 4. Flower cluster with round 5000 flowers

The Annual Global and Egyptian production of mangoes

Globally, mango production is increasing. In 2003, over 25 million tonnes of

Table1. Distribution of world production during four years by continents (million

Continent 2000 2001 2002 2003

Figure 6. Distribution mango in 6 major provinces in Egypt.

Recently, according to the FAO organization (www.fao.com) (Figures 7 and 8).

Figure 8. Total production 103 t during four years (2000 to 2004)

Table 3. Mango imports into European countries (source www.fao.org)

2000 2001 2002 2003

1. Overall the shelf-life is short.

3. Mango is sensitive to storage temperatures below 12oC.

Farm Harvesting Handling

Aim of this thesis

temperate or warm-temperate regions. This study will be restricted to investigating

Scope of the thesis

Chilling stress during storage leads to typical visible symptoms in various

Chilling injury and the balance between active

Loay Arafat, Jeremy Harbinson and Olaf van Kooten

1. Chilling injury symptoms in Mango

2. Chilling injury and its mechanism: the bilayer membrane

solidification and phase separation of the membranes also disrupts normal

Stress (e.g. Low Temperature)

Chilling injury symptoms

3. Chilling injury and its mechanism: Oxidative stress

In addition to the role for membrane dysfunction at low temperatures, it is

3.1. The mitochondria and active oxygen species formation

3.2. Chloroplast physiology as a source of active oxygen species

In chloroplasts AOS are generated from at least three sites in chloroplasts

under conditions where NADP is limiting. Photo-activated, excited chlorophyll

3.3. Cell wall and AOS

reactions, for example, the cross linking of phenylpropanoid precursors by

3.4. AOS from the plasma membrane

Superoxide-generating NAD(P)H oxidase activity has been clearly identified in

4. Oxidative stress: sensitivity, tolerance and occurrence

H2O2 generated from cell wall

TCA Cyt.c  2H2O

Factors determining sensitivity to, or occurrence of, oxidative stress

In addition to internal factors, such as antioxidant pool sizes, the physical

4.1. Harvest maturity

4.2. Storage temperature

Control of storage temperature is used to optimise or maintain quality

4.3. Water status

Hodges et al (2004) reported that there was a relationship between water

Physical abrasions, high or low processing temperatures, and product

4.6. Ripening and AOS

Ripening is associated with rapid changes in cellular components, an increase in

TCA Cyt.c 2H2O