Journal of Experimental Botany, Vol. 48, No. 308, pp. 769-778, March 1997
Journal of
Experimental
Botany
Analysis of physiological and molecular changes in melon
(Cucumis melo L.) varieties with different rates of
ripening
Alexandros Aggelis 13 , Isaac John1'2, and Don Grierson1'4
1
Department of Physiology and Environmental Science, The University of Nottingham, Sutton Bonington
Campus, Loughborough LE12 5RD, UK
2
Biology Department, The University of Michigan, Ann Arbor, Ml 48109-1043, USA
Received 28 June 1996; Accepted 10 October 1996
Abstract
Introduction
Seven melon varieties (Alpha, Delada, Marygold, Sirio,
Topper, Tornado, and Viva) known to exhibit differences in their ripening behaviour were used in this
study. The expression of mRNAs for ACC oxidase
(MEL1) and phytoene synthase (MEL5), required for
synthesis of ethylene and carotenoids, respectively,
and two ripening-related cDNAs (MEL2 and MEL7), of
unknown function, was examined and correlated with
the development of colour and softening of fruits. The
MEL2 and MEL7 mRNAs were present and accumulated in all varieties, indicating their importance in
melon fruit ripening. The fruits of Delada and Marygold
did not show any change in the colour of the flesh
even at 50 daa (days after anthesis). All other varieties
changed colour from green to orange between 25-30
daa. The phytoene synthase mRNA levels in most varieties seemed to be unrelated to change in fruit flesh
colour. The firmness of all the fruits was reduced
significantly between 25 and 40 daa. The expression
of ACC oxidase mRNA showed the most variation
among the different varities and was delayed in Sirio
and undetectable in Marygold fruits even at 40 daa.
Varieties with delayed expression of ACC oxidase
mRNAs after anthesis also showed delayed softening
during ripening. The prospects of genetic engineering and breeding for melon fruits with improved
quality characteristics and extended storage life are
discussed.
Melons (Cucumis melo L.) are commercially important
fruits, but their ripening has been relatively poorly studied
compared to other fruits such as tomatoes, avocadoes or
apples. A large number of diverse melon cultivars are
available that exhibit variation in ripening characteristics.
Early and late harvesting varieties are known for many
fruits, but in melons a selection can also be made according to fruit colour, shape or sweetness. There is also
variation in the respiratory climacteric, which is probably
a variety-dependent characteristic of melon (Hadfield
et al., 1995; Nukaya et al., 1986). All these differences
offer a vast gene pool that can be biochemically and
genetically exploited.
The response of melon fruits to endogenous production
and exogenously supplied ethylene has been studied biochemically. This has focused mostly on the accumulation
of sugars because sweetness is the most characteristic
attribute of melon fruits (Lingle and Dunlap, 1987).
Softening of melon fruits during ripening involves modifications of cell walls, but the mechanisms and the
enzymes involved are not well characterized (McCollum
et al., 1989; Ranwala et al., 1992). Studies at the molecular
level of specific genes involved in ripening are scarce.
Classical breeding has made many improvements in
fruit quality mainly by selecting varieties with disease
resistance characteristics. It is worth noting that these
improvements have been made in the absence of detailed
knowledge about the genetical background of the plants
and the physiological interactions involved in the pathogenesis process. Genetic transformation of melons is now
Key words: Cucumis melo, colour development, melon
varieties, ripening genes, softening.
3
4
Present address: Institute of Viticulture and Vegetable Crops, National Agricultural Research Foundation, 71110 Heraklion, Crete, Greece.
To whom correspondence should be addressed. Fax: +44 115 951 6334. E-mail: [email protected]
6 Oxford University Press 1997
770 Aggelis et al.
possible (Dong et al., 1991; Ayub et al., 1995) and this
method is likely to be used for improving melon, as it
has been for other crops (De Block, 1993). However,
genetic transformation and classical breeding will be more
efficient in improving the quality of melon fruits and
extend storage life if there is better knowledge of the
ripening process and the genes involved.
Previously, melon cDNA clones encoding unknown
polypeptides, plus those encoding enzymes involved in
the synthesis of ethylene and carotenoids have been
characterized (Balague et al., 1993; Karvouni et al., 1995;
Aggelis et al., unpublished data). In order to contribute
to the understanding of melon ripening, an attempt was
made in this study to correlate the mRNA expression of
ripening-related genes with the ripening phenotypes of
different melon varieties, known to exhibit normal or
slow-ripening and long shelf-life characteristics after harvesting. The expression of four ripening-related mRNAs
(MEL1, MEL2, MEL5, and MEL7) for cDNAs isolated
from a ripe fruit cDNA library was examined at different
ripening stages in parallel with physiological changes.
probe on the fruit flesh 1.5 cm below the epidermis. The a value
(bluish-green/red-purple hue component) was plotted against
the age of the fruits after anthesis. The data presented in Fig. 1
are means of four measurements taken at four different positions
for each fruit.
Texture measurements
For the texture measurements, a cylindrical sample of fruit
tissue 2 cm in length and 15 mm diameter was removed using a
metallic cork borer, starting from the epidermis inwards to the
seed cavity. The cylinder was compressed with a 12 mm
diameter probe against a metallic base. The force, in Newton
(N), required to suppress each sample was plotted against the
respective distance in mm, till the sample collapsed, using a
TA-XT2 Texture Analyser (Stable Micro Systems). The slope
of the curve (Nmm" 1 ) indicated the firmness of the tissue
examined. The data plotted in Fig. 2 are the mean of three
measurements.
Ripening-related genes
Materials and methods
Four cDNA clones (MEL1, MEL2, MEL5, MEL7) for
mRNAs that increased their expression during ripening of
melon fruits were used. MEL1 encodes ACC-oxidase (Balague
el al, 1993) and MEL5 encodes phytoene synthase (Karvouni
el al., 1995). The MEL2 and MEL7 cDNA clones have
unknown functions during melon ripening (Aggelis et al.,
unpublished data).
Plant material
Northern analysis
The seeds of slow-ripening varieties of melon were obtained
from different sources. Dr Rob Dirks (Nunhems Zaden BV,
Haelen, Holland) provided the Topper, Delada (Galia Delada),
and Viva varieties. Topper has been developed and characterized
as a long shelf-life variety. Viva has a normal shelf-life. Delada
has a longer shelf-life than Viva, but it has not been developed
for those characteristics as has Topper. The Sirio, Alpha and
Tornado varieties were provided by Mr F Ignart (Tezier
Breeding Institute, Velence, France). Sirio and Tornado were
characterized as slow-ripening varieties while Alpha shows
normal ripening. The seeds of Marygold Casaba variety
provided by Professor Timothy Ng (Department of
Horticulture, University of Maryland, USA), have been characterized as very slow ripening. Marygold belongs to the Inodorus
group while all other varieties used in this study probably
belong to the Cantaloupensis group (Munger and Robinson,
1991; Pharr and Hubbard, 1994).
Seeds of all varieties were surface-sterilized in a 50% sodium
hypochlorite solution for 20 min and thoroughly rinsed with
distilled water. Seeds were sown at the same time in Fisons M2
compost in 5 1 pots and grown in glasshouses under 16 h of
light. The plants were supplemented with Hoagland's solution
every second day (Hoagland and Arnon, 1950). Freshly opened
female flowers were hand-pollinated and tagged to identify fruit
of known age (McGlasson and Pratt, 1963). One fruit per plant
was allowed to develop. Fruits were harvested every 5 d starting
from 15 d after anthesis (daa) until the overripe stage or until
abscission occurred. The mesocarp tissue was separated from
the contents of the seed cavity and epidermis, cut into small
pieces, frozen in liquid nitrogen, stored at — 70 °C and used for
Northern analysis.
Total RNA from fruit samples was extracted using the method
described by Smith et al. (1986) and Northern blot analysis
was carried out as described by John el al. (1995). The
membranes were exposed for autoradiography at — 70 °C using
intensifying screens. In addition to autoradiography, signal
intensity on the membranes was quantified directly using an
AMBIS 4000 radioanalytical imaging detector and analysed
using AMBIS QuantProbe version 4 software.
Colour measurements
The firmness of all the fruits was reduced significantly
between 25 and 40 daa, but the rate of softening differed
between the varieties (Fig. 2). There was a difference of
The fruits were cut longitudinally and the measurements were
taken with a Chroma meter (Minolta CR-200), by placing the
Results
Colour changes
The colour of the fruit flesh was measured in fruits of
each variety (Fig. 1). The fruits of the Delada and
Marygold varieties did not show any change in flesh
colour even at 50 daa, but remained green-white throughout ripening of the fruits. The Alpha, Tornado and Sirio
fruits changed colour from green to orange between 25
and 30 daa. Topper and Viva fruits changed from green
to orange between 30 and 35 daa. The colour of the skin
of all varieties changed from green to brown/yellow
during ripening except the Marygold fruits, which
turned from green to bright yellow at 35 daa (data
not shown).
Texture changes
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Fig. 1. Colour changes in different melon fruit varieties. The colour of the fruit flesh was measured and the a hue component was plotted against
the age ot the truits. Negative values indicate green fruits and positive values orange fruits
5-10 d in the time required for the fruits of different
varieties to get soft for harvesting. The firmness value of
5 Nmm" 1 corresponded with the time when the fruits
were soft and juicy and was used to compare the rate of
softening in different varieties. The Viva variety had the
firmest fruit (15 N mm" 1 ) early in development. Tornado
fruits were the first to start softening, at 20 daa. Marygold
and Sirio fruits had the slowest rate of softening, reaching
5 N mm "' at 40 and 45 daa, respectively. Alpha, Tornado
and Delada fruits reached 5 N mm ' at 35 daa while
Topper and Viva were softer than 5 Nmm" 1 before
35 daa.
Expression of MEL2 and MEL7 mRNAs
MEL2 mRNA was expressed in all varieties and its
accumulation increased during ripening. In Viva, Tornado
and Sirio it showed a maximum expression at 35 daa and
772
Aggelis et al.
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Fig. 2. Texture changes in various melon varieties. The firmness (required force per mm of deformation) of the fruit flesh in Newton ( N ) m m
was measured and plotted against their age.
then decreased to 70-80% of the maximum. In the other
four varieties it continued accumulating till 40 daa. In
Viva, Topper, Alpha, and Delada fruits it was not detected
before 30 daa (Fig. 3). The MEL7 mRNA in Topper
became detectable at 30 daa while in all other varieties it
was present at all stages but increased during ripening
(Fig. 4). In Viva, Tornado, Delada, Sirio, and Marygold
its levels continued to accumulate till 40 daa. In Topper
and Alpha MEL7 mRNA was maximum at 35 daa and
then decreased to 60-70% of the maximum at 40 daa
(Fig. 4).
Expression ofphytoene synthase mRNA
The expression pattern of MEL5 mRNA, which encodes
phytoene synthase, was examined. The mRNA was
detected at all stages in every variety, but mRNA levels
were always lower as compared to other ripening-related
Melon genes and ripening changes
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Fig. 3. Expression of MEL2 mRNA in different melon varieties. Total RNA from ripening melon fruits was electrophoresed in 1% agarose gels,
blotted on to nylon membranes and hybridized with MEL2 probe. The lower panels were derived after quantifying the radioactivity hybridized to
the membranes and the results are shown as a percentage of the maximum signal. The 100% CPM for Viva, Topper, Tornado, Alpha, Delada,
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Melon genes and ripening changes
mRNAs examined. It did not show any major change in
expression during ripening although fluctuations in some
varieties were observed. In Viva, Alpha and Delada the
MEL5 mRNA expression was unchanged during ripening.
In Topper MEL5 mRNA continued accumulation till 40
daa while in Marygold the amounts decreased during
ripening. In Tornado and Sirio fruits the mRNA accumulated till 35 daa and then decreased to 30-^0% of the
maximum thereafter (Fig. 5).
Expression of ACC oxidase mRNA
The expression of MEL1 mRNA, which encodes ACCoxidase, was determined in all melon varieties and showed
considerable variation. There was no detectable MEL1
mRNA in the Marygold fruits up to 40 daa. In Alpha
and Sirio fruits it was detected only at 40 daa. In Viva,
Topper and Delada fruits it was present from 30 daa and
accumulated till 40 daa. In Tornado the MEL1 mRNA
showed maximum expression at 35 daa and decreased
slightly at 40 daa (Fig. 6).
Discussion
A high percentage of perishable fruits is lost by damage
during transport and storage. Some of these losses can
be prevented through better understanding of the postharvest physiology of these fruits and by exploiting knowledge of the ripening process in slow ripening melon
cultivars. Since all melon varieties used in this study were
grown under the same conditions and at the same
location, the variations can be attributed to the genetic
differences and not environmental influences.
Two of the slow-ripening varieties (Marygold and
Delada) developed fruits with green flesh. The colour of
the flesh remained unchanged even when the fruit was
overripe (Fig. 1). Instead of degreening, the intensity of
the green colour seemed to increase during ripening, indicating the presence and perhaps the synthesis of chlorophylls. Although the fruits appeared to ripen normally,
subjectively they had inferior taste compared to the other
varieties with orange flesh. In the fruits that developed
orange flesh both colour and firmness changed rapidly
during ripening. The loss of the green colour, indicated by
the increasing a values, was followed by a dramatic
decrease of firmness of all fruits, except in the Sirio variety
where this decrease was delayed. The change of colour
started always from the tissue around the seed cavity
following, presumably, the production of ethylene. At 35
daa the orange colour had spread to all the mesocarp
tissue of the fruits of all the orange flesh varieties.
The softening patterns also showed some genotypic
variation (Fig. 2). In the varieties that exhibited the
slowest rates of softening (Marygold and Sirio), there
was also a delay in the initiation of the process. Once
softening was started the fruits of all varieties softened
continuously. The texture of fruits with firmness values
l
775
lower than 5 N mm ~ was considered as not commercially
acceptable. The long shelf-life genotypes (Topper and
Tornado) did not show remarkable difference in the
softening of the mesocarp tissue compared with the
normal ripening (Alpha and Viva) varieties (Fig. 2). It is
possible that differences existing in the softening of the
fruit exocarp and epidermal tissues might be responsible
for the long shelf-life phenotype. These possible differences could be examined by studying the behaviour of
intact fruits in compressibility tests.
There was an attempt to correlate phenotypic and
physiological changes with mRNA expression of ripeningrelated genes. Expression of ACC oxidase (MEL1),
phytoene synthase (MEL5), MEL2 and MEL7 mRNAs
was determined at different ripening stages. There was an
increase in the levels of MEL2 and MEL7 mRNAs in all
melon varieties during ripening (Figs 3, 4) although there
was delayed detection of MEL2 and MEL7 mRNAs in
Topper as compared to all other varieties.
In tomato, an increase in phytoene synthase mRNA
during ripening has been shown to be required for the
production of carotenoids (Bird et al., 1991). The constitutive expression of the phytoene synthase (MEL5) mRNA
in the green flesh varieties Delada and Marygold suggested
that its expression does not necessarily lead to the appearance of colour change in the fruits (Fig. 5). This is in
agreement with the observation that MEL5 mRNA levels
remained unchanged or decreased during the colour change
of the fruits in Alpha and Viva varieties, respectively. The
possibility of regulatory mechanisms in the subsequent
steps of the carotenoid biosynthesis pathway and the
necessity for regulating chlorophyll degradation implies
the coordinated expression of more than one gene for
melon fruit flesh coloration. In tomato, there are known
to be at least two genes for phytoene synthase (Fray and
Grierson, 1993) and genomic Southern analysis suggests
that there are also multiple genes in melon (Karvouni
et al., 1995). The possibility exists, therefore, that, as in
tomato, there are different melon phytoene synthase genes
involved in synthesis of carotenoids in green tissues, as
well in ripening tissues undergoing colour change.
The ACC oxidase mRNA (MEL1) showed the largest
variation among different varieties. In Viva, Topper and
Tornado fruits the change in colour from green to orange
occurred in parallel with the detection of the ACC oxidase
mRNA in the mesocarp tissue (Fig. 6). In Alpha and Sirio
varieties the colour change occurred at least 10 d before
the detection of ACC oxidase mRNA, whereas in Delada,
appearance of ACC oxidase mRNA did not affect the
maintenance of the green colour of the fruit flesh. There
was no ACC oxidase mRNA detected in the Marygold
variety till 40 daa, the latest stage examined. Although the
gene(s) might be expressed later during ripening, the lack
of colour change and the slow rate of softening suggest a
modified ripening process in this genotype that is related
to altered ACC oxidase gene expression.
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Fig. 5. Expression pattern of MEL5 during ripening in melon varieties. The RNA samples and quantitation of the radioactivity are descnbed in
the legend to Fig. 3. The membranes were hybridized with MEL5 probe.The 100% CPM for Viva, Topper, Tornado, Alpha, Delada, Sirio, and
Marygold were 5.9, 2.8, 1.7, 8.4, 19.8, 18.6, and 8.2,respectively.Each sample was counted for 10-16 h.
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Melon genes and ripening changes
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Fig. 6. Expression of MELl mRNA during ripening in melon varieties. The RNA samples and quantitation of the radioactivity are described in
the legend to Fig. 3. The membranes were hybridized with MELl probe. The 100% CPM for Viva, Topper, Tornado, Alpha, Delada, Sirio, and
Marygold were 224.7, 197.6, 44.4, 77.1, 218.2, 59.4, and 8.3, respectively. Each sample was counted for 10-16 h.
778
Aggelis et al.
Recently, reduction in expression of ACC oxidase
mRNA in melon has been achieved by genetic modification, using an antisense gene (Ayub et al., 1996). This
inhibited loss of firmness during ripening. No clear correlation was found between the softening of the flesh and
the detection of ACC oxidase mRNAs accumulation
in melon varieties in the experiments. In Alpha and
Marygold varieties softening of the fruit flesh proceeded
without any detectable accumulation of ACC oxidase
mRNA. In Tornado variety fruits, softening started in
the absence of detectable ACC oxidase mRNA expression
and the rate of softening was not affected when the ACC
oxidase mRNA was highly expressed at 30 daa. In Topper,
Viva, Sirio, and Delada varieties, softening seemed to
occur in parallel with the expression of ACC oxidase.
With the exception of ACC oxidase, all the mRNAs
for ripening-related genes were present in all varieties.
Their levels continued to increase even at 40 daa in some
varieties, suggesting that these ripening-related genes are
important for the progress of ripening. Additional work
is needed to determine the function of MEL2 and MEL7
and to identify additional ripening-related genes from
melon. Further experiments examining the ethylene production and the expression of ACC oxidase in later stages
of ripening are necessary in order to resolve the question
whether the Marygold variety is an ethylene deficient
mutant and whether this characteristic is associated with
the indorous background in the genotype. Clarification of
this issue may help in designing strategies for genetic
modification of melon cultivars.
Molecular cloning of ripening-related cDNAs has
enabled the identification of novel plant genes encoding
enzymes involved in cell wall texture change, carotenoid
biosynthesis, ethylene synthesis, and the identification of
gene control regions involved in fruit-specific, ripeningspecific, and ethylene-regulated gene expression (Grierson
and Schuch, 1993). Antisense and partial sense gene gene
techniques have been used to generate genetically modified
plant lines in which specific genes have been inactivated
during ripening (Ayub et al., 1996; Grierson and Schuch,
1993). Genetic transformation of melons is now possible
and genes involved in the accumulation of aromatic
volatiles, sugars and colour, as well as the rate of overripening and spoilage can potentially be manipulated, to
generate commercial lines with desirable characteristics.
Acknowledgements
Alex Aggelis was in receipt of EU Training and Mobility for
Researchers Bursary. Part of this work was supported by an
EU ECLAIR grant (Contract AGRE 015).
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