PHYSIOLOGIA PLANTARUM 110: 248–255.

2000 Copyright © Physiologia Plantarum 2000

Printed in Ireland —all rights reser6ed ISSN 0031-9317

Spatial-resolved analysis of histological and biochemical alterations

induced by water-soaking in melon fruit

C. du Chateneta,b, A. Latchéb, E. Olmosc, B. Rantya, M. Charpenteaua, R. Ranjevaa, J. C. Pechb and A. Grazianaa,*

a
Laboratoire CNRS/UPS UMR n°5546 de l’Uni6ersité Paul Sabatier, Pôle de Biotechnologie Végétale, 24, chemin de Borde Rouge, BP 17
Auze6ille, F-31326 Castanet-Tolosan, France
b
ENSAT, UMR INRA n°990, Pôle de Biotechnologie Végétale, 24, chemin de Borde Rouge, BP 107 Auze6ille, F-31326 Castanet-Tolosan,
France
c
CSIC-CEBAS A6enida de la Fama, E-30080 Murcia, Spain
*Corresponding author, e-mail: graziana@cict.fr
Received 10 November 1999; revised 16 February 2000

Water-soaking, a physiological disorder characterised by a
glassy texture of the flesh, depreciates greatly the commercial
quality of early-season Charentais cantaloupe melons (Cu-
cumis melo L. cv. Talma). Although it is accepted that the
genotype and a number of physiological and environmental
factors play a role in the development of the syndrome, the
intimate mechanisms responsible for water-soaking remain
unknown. We report here on an integrated study of the
development of water-soaking in fruit. Using nuclear magnetic
resonance (NMR) imaging, we have shown that water mobil-
ity increased in the diseased tissues. Alteration of the cell wall
and the presence of large intercellular spaces were correlated
with a severe depletion of cell wall calcium. Water-soaking
developed during the late stages of fruit ripening, but no
correlation was found with ethylene biosynthesis. Thus, fruits
in which ethylene action was blocked by 1-methylcyclopropene
remained sensitive to water-soaking. Moreover, the expression
of two genes encoding key enzymes in ethylene biosynthesis
remained unchanged in response to water-soaking. The major
changes observed concerned a protein implicated in calcium
signalling processes. While the amount of total calmodulin,
the ubiquitous calcium binding protein, was not modified, a
particular calmodulin-binding protein (CaM-BP) was absent
in water-soaked but not in sound mature tissues. This CaM-
BP may be a marker or a determinant of this physiological
disorder.

Introduction
Water-soaking is an economically important metabolic dis-
order which causes considerable loss in early season produc-
tion of melon. It is characterised by an alteration of the flesh
texture which becomes dark and glassy, and by an early
over-maturity of the fruit. It is thought that calcium and
ethylene may play a role in promoting the disorder as these
signals are known to be involved in many processes includ-
ing fruit ripening and organ senescence (Sams et al. 1993,
Deikman et al. 1998). However no critical assessment of this
hypothesis has been performed to date.
Ethylene synthesis is induced either at specific stages
during plant development or by biotic and abiotic stress
(Fluhr and Matoo 1996). It has been shown that this
stress-stimulated ethylene production serves as a secondary
signal for the induction of specific genes (eg a pathogenesis-
related chitinase gene) and has important physiological con-
sequences such as on phenotypic plasticity (Raz and Fluhr
1992, Kacperska 1997, Kathiresan et al. 1998). The onset of
ethylene production is the result of the concomitant increase
in the activities of ACC synthase and ACC oxidase (ACS
and ACO). In melon, a gene encoding ACS (ME-ACS1 ) has
been isolated and shown to be preferentially expressed in
response to wounding (Miki et al. 1995). Moreover, three
genes encoding ACO homologues have been isolated from
melon and were found to be expressed differentially. One of
the three genes (CM-ACO1 ) is up-regulated in response to
biotic and abiotic stress such as wounding, salt stress or
drought (Bouquin et al. 1997).
In addition to ethylene, it has been suggested that calcium
may be implicated in water-soaking. Calcium has long been

Abbre6iations – ACC, 1-aminocyclopropane-1-carboxylic acid; CaM, calmodulin; CaM-BP, calmodulin binding protein; CM-ACO, Cucumis
melo ACC oxidase; CM-ACS, Cucumis melo ACC synthase; 1-MCP, 1-methylcyclopropene; ST, sound tissues; WST, water-soaked tissues.

248 Physiol. Plant. 110, 2000

recognised as an essential plant nutrient involved in a number ms, and for multi slice multi echo (MSME), TE =25.5 ms (8
of physiological processes and in preventing physiological echoes), TR= 3000 ms. The field of view was 15 cm, the slice
disorders in fruit and plant tissues during growth and devel- was 20 mm thick, and the image matrix, 128×128.
opment (Fry 1986, Dawson et al. 1993, Stow 1993, Lester
1996). It has been hypothesised that the development of
water-soaking in the melon is related to a deficiency in calcium Scanning electron microscopy and X-ray microanalysis
(Bernadac et al. 1996, Jean-Baptiste et al. 1999). Moreover,
Tissue ultrastructure and mineral element contents were
it has been shown that calcium acts as a second messenger
determined by electron microscopy coupled with X-ray anal-
in plant cell signalling (Knight et al. 1997, Mazars et al. 1997,
ysis according to Olmos and Hellin (1998). Mineral elements
Frohnmeyer et al. 1998, Gong et al. 1998, Long and Jenkins
were individually identified and quantified automatically. For
1998) generally via interactions with calmodulin, a small
each ion of interest, the apparent concentration was deter-
calcium binding protein. Changes in internal cellular calcium
mined in three independent experiments according to the
concentration may thus regulate the activity of many enzymes
equation (AC = intensity element A /intensity element
through the formation of functional complexes with calcium-
Astandard × weight percent (Wt%) of the element Astandard).
activated CaM, leading to changes in cell organisation and
gene expression (Zielinsky 1998).
In the present paper, we have combined physical (NMR
RNA extraction and reverse transcriptase-polymerase chain
imaging), biochemical (ethylene production, calmodulin-
reaction (RT-PCR)
binding proteins activity), molecular (expression of calmod-
ulin and genes encoding enzymes involved in ethylene Total RNAs were isolated from melon fruit according to
biosynthesis) and ultrastructural (SEM coupled to X-ray Balagué et al. (1993) and cDNAs synthesised as described by
analysis) methods to address the relationship between water Lasserre et al. (1996), using 20 mg of total RNA, followed by
soaking, ethylene and calcium. The non invasive, non destruc- PCR with appropriate primers. Primers were chosen from the
tive attributes of 1H MRI, and its ability to provide highly 5% and 3% regions of the genes of interest. Primers amplified
resolved spatial information concerning the distribution and full length cDNAs for ACO and CaM, and a 3% internal
magnetic environment of water in soft tissues (Clark et al. fragment for ACS1 (Miki et al. 1995).
1997) has been used to detect water-soaking. We have been
5%-GGATGGATTTGAGGAAGCTACTA-3% (CM-ACS1
able to show that although being structurally disorganised,
5%),
water-soaked tissues were not affected in terms of ethylene
5%-GCTCATCCTCATTAAGTCTTGGC-3% (CM-ACS1
production and calmodulin gene expression, suggesting that
3%),
they remain metabolically active. In contrast, the tissue
5%-GAATTCATGGCTGTCTTTCCTATCATC-3% (CM-
texture was greatly affected and a calmodulin-binding protein
ACO1 5%),
was not detected in water-soaked tissues.
5%-CTCGAGTTATGCTGTTGCCATTGGAC-3% (CM-
ACO1 3%),
5%-CAGCTCACCGACGATCAGATC-3% (CM-CaM 5%),
Materials and methods 5%-TCCTCGTAGTTAATCTGGCC-3% (CM-CaM 3%).
Plant material All RT-PCR reactions were carried out in triplicate on two
independent RNA extractions. The accumulation of mRNA
Melon plants (Cucumis melo L. cv. Talma), provided by
was estimated by RT-PCR using the constitutively expressed
INRA (Domaine de la Grande Ferrade, Bordeaux, France)
CMe-56.10 gene (Pech et al. 1999). RT-PCR analysis was
were grown in soil-less culture in a glasshouse, with a
chosen for the expression studies as it is more amenable to
day/night temperature of 20/18°C. The ripening stages of the
the study of low copy number transcripts such as ASC1.
fruit were assessed by measuring ethylene production (Ayub
et al. 1996) and the development of water-soaking was
monitored by NMR imaging. After harvest, fruits were stored
Southern blot analysis
at 14°C. Pedoncular (sound tissues) and distal (water-soaked
tissues) parts of the preclimacteric fruit were frozen in liquid PCR products were resolved by electrophoresis on a 0.8 or
nitrogen and stored at −80°C. For inhibiting ethylene action, a 1.4% (w/v) agarose gel, transferred to nitrocellulose mem-
preclimacteric fruits were treated overnight with 1 ml l − 1 of branes and hybridised at 65°C with a [a-32P] dCTP random
1-methylcyclopropene (1-MCP), harvested and stored at primed cDNA insert (ACS or ACO or CaM). Membranes
14°C. Ethylene production was measured and fruit samples were washed at high stringency in 2 × standard sodium citrate
were then frozen. buffer (SSC), 1% (w/v) SDS at 55°C for 15 min. Membranes
were autoradiographed using Amersham Hyperfilm-MP with
intensifying screen at −80°C.
NMR imaging
The NMR images were taken with a TOMIKON R23 whole
Production of GST-CaM fusion protein in E. coli
body imaging system (0.23 T, 10 MHz proton frequency). The
parameters for gradient echo fast imaging (GEFI) were as A GST-CaM fusion protein was produced in order to
follows: echo time (TE) =13 ms; recuperation time (TR) =50 prepare a 33P-CaM protein probe, and to allow CaM-BP

Physiol. Plant. 110, 2000 249

purification by affinity chromatography. A synthetic VUC-1
CaM gene (Roberts et al. 1985) was fused to a sequence
which encodes a peptide carrying a phosphorylation site and
this modified CaM was expressed as a fusion protein with
the glutathione S-transferase (GST) in the pGEX system
(Pharmacia). The GST-fusion protein was expressed in E.
coli according to the manufacturer’s recommendations. The
GST-fusion proteins in E. coli crude extracts were purified
by affinity chromatography with Glutathione Sepharose 4B
beads (Pharmacia).
Extraction of proteins from melon and purification of
CaM-BP
All procedures were carried out at 4°C. Melon fruit tissue (5
g) was ground in a mortar with 10 ml buffer (100 mM
Tris-HCl, pH 7.5; 5 mM EDTA pH 8; 5 mM EGTA; 14
mM b-mercaptoethanol; 1 mM PMSF; 4 mM leupeptine
and 5% [w/v] PVPP). The homogenate was centrifuged for
15 min at 20000 g to yield a supernatant which was termed
the crude extract. Protein concentrations were determined
using the BCA protein assay (Pierce). The CaM-BP in crude
extracts was purified by GST-CaM affinity chromatography.
The extract was loaded on an affinity column (100 ml, 50 mg
CaM) equilibrated with Tween Tris-buffered saline (TTBS)
supplemented with 1 mM CaCl2. After extensive washing
with TTBS containing CaCl2, CaM-BP was eluted (fractions
of 100 ml) by TTBS buffer supplemented with 5 mM EGTA.
Proteins are separated by SDS-PAGE and the protein bands
were visualized by silver nitrate coloration.
Immunodetection of calmodulin
Proteins were separated by SDS-PAGE with 15% (w/v)
acrylamide gels according to the method of Laemmli (1970).
Proteins were electrotransferred onto PVDF membranes
(PolySreen™; NEF-1000) by the method of Hulen et al.
(1991), and cross-linked to the membrane by a 45-min
incubation in 0.2% (w/v) glutaraldehyde in Phosphate-
buffered saline (PBS). The membrane was blocked for 2 h at
room temperature with 5% (w/v) non-fat dry milk in PBS.
After washing, polyclonal IgY (1:500 in PBS) raised against
spinach calmodulin (Sigma) were applied to the membrane,
250
and incubated 1 h 30 at 37°C. After washing, the membrane
was incubated 1 h at 37°C with secondary antibodies raised
against chicken IgY (1:60000 in PBS) coupled to
horseradish peroxidase. The signal was detected by chemilu-
minescence (kit ECL + Plus, Amersham).
33
P-labelling of calmodulin and detection of CaM-BP by
overlays
An aliquot of gluthathione Sepharose 4B containing the
fusion protein (100 mg of calmodulin) adsorbed on beads,
was incubated (2 h at room temperature) with 18.5 MBq
33
gP-ATP and 500 units of protein kinase A catalytic sub-
unit (Sigma). Residual ATP was removed by successive
centrifugation. The labelled protein was cleaved by
thrombin digestion (1 unit) in 50 mM Tris-HCl, pH 8; 150
mM NaCl and 3.5 mM CaCl2, 30 min at 25°C, and after
centrifugation,33gP-CaM was recovered in the supernatant.
33
gP-CaM was used to detect target CaM-BPs by overlays.
Crude proteins extracts of melon were separated by SDS-
PAGE and electrotransferred to nitrocellulose membranes.
Membranes were incubated 60 min at room temperature in
TTBS buffer containing 5% non fat dry milk, and washed 10
min in TTBS buffer containing either 1 mM CaCl2 ( Buffer
A), or 5 mM EGTA (Buffer B). Membranes were incubated
overnight at 20°C with 33gP-CaM in TTBS buffer contain-
ing 1% non fat dry milk and either 1 mM CaCl2 (Buffer A)
or 5 mM EGTA (Buffer B). Membranes were washed three
times in the respective solutions without CaM, dried, and
exposed to Kodak Biomax™ – MR scientific imaging film.
Calcineurin, a 60-kDa Ca2 + -dependent CaM-binding
protein was used as a positive control.
Results
Structural and ultrastructural characterisation of
water-soaking
The non-invasive nature of MRI makes it suitable for
studies of water-soaking, where external symptoms are not
visible until after injury development is well advanced. It
was used to monitor water-soaking development in a non-
Fig. 1. Tissue structure of
water-soaked melon fruit. A.
Photograph of a longitudinal
water-soaked melon section. B.
NMR imaging of the same fruit.
NMR images were obtained by
gradient echo fast imaging. Zone 1
indicates the sound pedoncular part
(control area) and zone 2, the
water-soaked distal part of the fruit.
Physiol. Plant. 110, 2000
Fig. 2. Scanning electron micrographs of sound (A,B) and water-soaked (C,D) melon tissues. Sound and water-soaked tissues correspond
respectively to zone 1 and zone 2 of Fig. 1. Arrows indicate intercellular spaces. A and C: × 25; B and D: ×300.
destructive manner by measuring physical parameters that
cannot be determined by other methods. Fig. 1A shows a
photograph of a water-soaked melon section and Fig. 1B
shows its corresponding GEFI NMR image before cutting
(whole and intact fruit). Comparison of an NMR image of
a whole fruit and a photograph of the same fruit after slicing
revealed a good agreement between areas of affected and
unaffected tissues. Symptoms appeared at first in the distal
part of the fruit. It appeared that the ‘normal’ (zone 1) and
‘water-soaked’ (zone 2) areas of a melon fruit could be
discriminated by visual observations (Fig. 1A) and by GEFI
sequence NMR imaging (Fig. 1B). Strong signal intensity
(bright areas) corresponding to high density could be ob-
served in the distal part of the fruit (zone 2). In MSME
sequences, a water-soaked area displayed a T1 hypersignal
(relaxation time) of 572920 ms as compared to 4609 20 ms
in a sound area (data not shown). High relaxation times T1
in water-soaked areas indicate that structural changes have
occurred in the tissue that allow higher proton mobility.
Scanning microscopy observations (Fig. 2) showed that,
whereas sound tissues (Fig. 2A,B) displayed normal shaped
and regular cells, water-soaked tissues (Fig. 2C,D) are col-
lapsed and have large intercellular spaces. Moreover, water-
soaked tissues showed very low turgescence, and cells had an
irregular form with few connections between them, suggest-
ing a disorganisation of the cell wall.
Physiol. Plant. 110, 2000
Ethylene production and expression of ethylene biosynthetic
genes in water-soaked tissues
Water-soaking is known to appear during the early stages of
the climacteric phase of fruit ripening, suggesting a possible
relationship with the upsurge of ethylene production. How-
ever we have not found any clear differences in the pattern
of ethylene evolution of water-soaked and sound fruit nei-
ther on the vine nor after detachment (data not shown). The
onset of climacteric ethylene production occurred 37 days
after pollination in both cases.
ACS and ACO transcript accumulation was determined
by RT-PCR. The RT-PCR products were visualised after
agarose gel electrophoresis and ethidium bromide staining
and their identity were confirmed by Southern blot hybridis-
ation. The evaluation of ACS and ACO transcript accumu-
lation indicated that the level of expression of ACS and
ACO was identical in sound and water-soaked tissues (Fig.
3).
In order to further substantiate the absence of correlation
between the development of water-soaking and ethylene
synthesis, preclimacteric melon fruit were treated with 1-
MCP, a potent inhibitor of ethylene perception (Sisler et al.
1995). Fig. 4 shows that ethylene production of 1-MCP-
treated melons was delayed by four days as a consequence
of the inhibition of the autocatalytic process of ethylene
production. Interestingly, the water-soaking syndrome ap-
251
Fig. 3. RT-PCR analysis of ACS and ACO1 expression. RT-PCR
was performed on total RNA extracts of sound (lane ST) and
water-soaked tissues (lane WST) of the same fruit. PCR conditions
were as described in Materials and methods. Experiments were
performed in triplicate using two different sets of RNA.
peared at the same time (35 days after pollination) in
both control and 1-MCP-treated fruit, indicating that the
development of water-soaking is ethylene-independent.
Calcium content of the cell wall
Little information is available on water-soaking, but cal-
cium has been proposed to be implicated in the disorder
(Bernadac et al. 1996). The ionic composition of the cell
wall middle lamella was analysed by scanning electron mi-
croscopy (SEM) coupled with X-ray microanalysis. The
results depicted in Fig. 5 showed that the apparent con-
centrations of Na + , Mg2 + , Cl − and K + were identical
in the two sets of samples but the apparent concentration
of cell wall calcium was 100-fold higher in the sound than
in the water-soaked tissues.
Calmodulin and calmodulin-binding proteins
Beside its structural role, calcium is involved as a second
messenger in cellular regulation. Calcium is not directly
active but requires protein sensors such as CaM, derived
from an evolutionary well-conserved protein family. It has
Fig. 4. Ethylene production of control (
) and 1-MCP-treated ( ) tissues of both the pedoncular and distal part of the fruit
melons. 1-MCP (1 ppm) was applied overnight before harvest
(day =0). Ethylene production by individual melons was deter-
mined by gas chromatography. The arrow indicates the onset of
water-soaking appearance.
252
Fig. 5. Apparent concentration of various ions in sound (hatched
bars) and water-soaked (grey bars) tissues. Determination of total
ions was performed by X-ray microanalysis in zones 1 and 2
described in Fig. 1.
been shown that some CaM genes are stress sensitive
(Braam et al. 1996, Polisensky and Braam 1996) and
therefore, we searched for possible variations of CaM
gene expression in affected tissues. To evaluate the poten-
tial role of calcium as a second messenger, we studied the
changes in CaM and CaM-dependent events in melon
fruit.
The expression of the CaM gene was monitored in
sound (Fig. 1, zone 1) and water-soaked samples (Fig. 1,
zone 2) by RT-PCR. Fig. 6A shows that CaM expression
was similar in sound and water-soaked tissues. In addi-
tion, the CaM protein was immunodetected in both type
of tissues and no difference was observed in the level of
this protein (Fig. 6B).
The putative role of CaM-dependent events in water-
soaking was addressed through a search for calmodulin-
binding proteins (CaM-BPs) in protein extracts from
water-soaked and sound tissues. For this purpose we used
a novel overlay assay using a 33gP-CaM probe (see Mate-
rials and methods) on crude extracts or proteins purified
by CaM affinity chromatography. Proteins of sound and
water-soaked tissues were analysed on SDS-PAGE and sil-
ver nitrate coloration (Fig. 7A) and the results show that
proteins from water-soaked tissues are not degraded (lane
2). Blots incubated with 33gP-CaM in the presence of
EGTA resulted in no detected band (data not shown)
ruling out non-specific interactions independent of Ca2 + .
Fig. 7B shows intense binding of CaM to the 60-kDa
calcineurin-positive control in the presence of calcium
(lane 5). In sound tissues (lanes ST), a single CaM-BP of
60 kDa was detected in both the crude extract (lane1) and
the purified fraction obtained by CaM affinity chromatog-
raphy (lane 2). This CaM-BP was absent in crude extracts
(lane 3) and affinity-purified fractions (lane 4) of water-
soaked tissues taken at the pre-climacteric stage. Observa-
tions made at earlier or later stages of ripening have also
indicated the absence of this 60-kDa CaM-BP protein in
water-soaked tissues while it was always detected in sound
(data not shown). The absence of the protein in water-
soaked tissues was independent of whether EGTA and
calcium were added to the extraction buffer.
Physiol. Plant. 110, 2000
Fig. 6. RT-PCR analysis of CaM
expression (A) and western blot analysis
of CaM expression (B). Analysis were
performed on extracts of sound (lane
ST) water-soaked tissues (lane WST) of
the same fruit. RT-PCR experiments
were done in triplicate using two
different sets of RNA. PCR and western
blot conditions are described in Materials
and methods.

Discussion production (Fig. 4) and mRNA levels of genes involved in

We have studied the relationship between water-soaking,
ethylene production and calcium during ripening of melon.
The results show that water-soaking appears during ripening
of the fruit and is not externally visible on whole fruit until
symptoms have reached very late stages of development. In
this study, the development of water-soaking of fruit tissues
was monitored, in a non destructive manner by NMR
imaging. NMR can detect physical parameters such as water
mobility and proton density which are not accessible by
other methods (Wang et al. 1988, Wang and Wang 1989,
Sonego et al. 1994, Clark et al. 1997). The NMR images
obtained can then be correlated to the texture of the tissues.
Our results indicate that water-soaking symptoms of melon
first appeared in the distal part of the fruit. A higher water
mobility associated with a higher density observed in water-
soaked areas as compared to sound tissues indicates an
increase in the level of free water i.e. water unbound to
cellular material.
In addition, scanning electron microscopy showed that
water-soaked tissues exhibit an abnormal morphology with
a disorganisation of the cell walls and an increase in intercel-
lular spaces. Similar symptoms have been observed in plant
tissues grown in vitro in which hyperhydricity was responsi-
ble for the translucency and ‘vitrification’ of the tissues
(Deberg et al. 1992, Sato et al. 1993). In this latter case, a
hypo-lignification was considered to be responsible for the
alteration of the mechanical properties of the cell wall
(Kevers and Gaspar 1985). Water-soaked tissues of melon
also exhibited a disruption of the cell wall that could reduce
cell turgor, alter water potential, increase water uptake and
finally result in an hyperhydricity of the tissues.
Since ethylene is known to participate in various physical
and environmental stress (English et al. 1995, Banga et al.
1996), its involvement in the development of water-soaking
in melon can be hypothesised. However, data on ethylene
ethylene biosynthesis (Fig. 3) lead to the conclusion that the
development water-soaking is ethylene-independent. One of
the strongest arguments for this conclusion is the dissocia-
tion of ethylene production and development of water-soak-
ing using the ethylene action inhibitor 1-MCP (Fig. 4).
Furthermore, melons expressing an antisense ACO gene
(Ayub et al. 1996) were able to develop water-soaking even
though their ethylene production was inhibited by more
than 99.5% (Guis, unpublished data). Taken together, these
data demonstrate that ethylene is not directly involved in
water-soaking.
A possible correlation between calcium and water-soaking
has been suggested by Bernadac et al. (1996), who found less
total calcium in the distal part of melon fruit. Calcium may
be involved essentially as a structural or nutritive element
but also may play a role as a second messenger. Calcium is
involved in maintaining the dynamic shape of the cell and
integrity of cellular membranes (Bangerth 1979, Paliyath et
al. 1984, Poovaiah 1988). Microscopic analysis showed the
presence of large intercellular spaces and a severe disorgani-
sation of the cell walls in water-soaked tissues. X-ray analy-
sis coupled to scanning microscopy, a versatile technique for
qualitative and quantitative estimation of chemical elements
from a well defined region of the melon sample, has demon-
strated that while the apparent concentrations of Mg2 + ,
Cl − , Na + and K + were similar in water-soaked compared
to sound areas, the calcium concentration was 100-fold
higher in the sound tissues. The very low levels of calcium
that we have measured in the water-soaked areas of the fruit
is not due to the distal localisation as Bernadac et al. (1999)
have shown that total calcium concentration in the flesh of
sound fruits was generally lower by only 3.5% in the distal
part than in the pedoncular part of the fruit. Thus the
alteration in calcium that we have measured is associated
with water-soaking. Calcium is known to stabilise cell walls
by establishing bridges between pectin polymers. A lower

Fig. 7. A. SDS-PAGE analysis of proteins

in crude extracts of sound (ST) and water
soaked tissues (WST). Proteins are colored
by silver nitrate. B. Overlay detection of
CaM binding proteins in extracts of
water-soaked and sound tissues of melon
fruit. Total proteins were separated on
SDS-PAGE, transferred to nitrocellulose
membranes and then incubated with a 33gP
CaM probe. Samples were prepared as
described in Materials and methods. Lanes
1 and 3 correspond to crude extracts (80
mg), lanes 2 and 4 to fractions eluted from
a CaM affinity column and lane 5 to a
control CaM-BP, calcineurin (20 ng).

Physiol. Plant. 110, 2000 253

calcium concentration in the cell wall could explain the
tissue disorganisation of water-soaked areas (Carpita and
Gibeaut 1993).
Concerning Ca sensing CaM, the similarity in CaM gene
expression and CaM protein levels between sound and
water-soaked tissues (Fig. 6) seems, at first sight, to exclude
a direct role of CaM in the disorder. However, using a novel
overlay technique, we have shown that a particular 60-kDa
CaM-BP is present in sound but not water-soaked tissues.
The detection of only one CaM-BP may be due to its high
abundance, with lesser abundant CaM-BP proteins perhaps
being present but not detectable using our techniques. A
limited number of CaM-BP have been identified in plants
but their functions remain mainly unknown. The finding
that a specific calmodulin-binding protein was detected only
in sound tissues and never in water-soaked tissues, strongly
suggests a role for alterations in calcium sensing in the
disorder.
Taken together our data demonstrate that although wa-
ter-soaked tissues are disorganised, the cells remain
metabolically active, as illustrated by the accumulation of
several mRNAs (ACS, ACO and CaM) and CaM protein.
Thus, the absence of the 60-kDa CaM-BP in water-soaked
tissues cannot be ascribed to a consequence of the deteriora-
tion of the tissue. We have shown here that this CaM-BP is
absent in water-soaked areas, but it remains to be deter-
mined whether its disappearance is a cause or consequence
of the disorder and whether it is involved in maintaining cell
integrity.
Acknowledgements – This work was supported by the E. U. grant
AIR 3.CT94.1961. and the EU (postdoctoral fellowship to EO). It
represents some of the research submitted by CD in the partial
fulfilment of the requirements for the doctorate degree. The authors
are grateful to F. Libes (AGROTEC, Agen, France) for his help in
NMR imaging and S. Thunot (INRA, Bordeaux, France) for the
melon cultures. We thank Julie Cullimore and Brian Jones for the
English revision of the manuscript.
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