Annals of Botany 88: 415±422, 2001
doi:10.1006/anbo.2001.1482, available online at http://www.idealibrary.com on
Histological and Physiological Characterization of Rind Breakdown of `Navelate' Sweet
Orange
M . A G U S T IÂ * {, V. A L M E L A {, M. J U A N{, F. A L F E R E Z {, F. R . TA D E O } and L . Z ACA R IÂ A S {
{Dept. ProduccioÂn Vegetal, Universidad PoliteÂcnica, Camino de Vera s/n, Valencia, 46022, Spain, {Instituto de
AgroquõÂmica y TecnologõÂa de Alimentos, CSIC, Apdo Postal 73, Burjasot 46100, Valencia, Spain and }Dept.
Citricultura y Otros Frutales, Instituto Valenciano de Investigaciones Agrarias, Moncada 46113, Valencia, Spain
Received: 2 April 2001 Returned for revision: 25 April 2001 Accepted: 16 May 2001
Rind breakdown of `Navelate' sweet orange is characterized by sunken colourless areas of the peel which develop into
reddish-brown, dry areas partially covering the exposed portion of the mature fruit. Sudden changes in relative
humidity at fruit colour break seem to be responsible for the natural development of this disorder, which begins at the
transitional zone of the ¯avedo-albedo and advances across the ¯avedo reaching the epidermis. A€ected cells have
reduced amounts of cytoplasm located in a central position and have twisted and squashed walls, forming areas of
collapsed cells amongst the healthy cells of the ¯avedo and albedo. Comparisons of healthy and damaged areas of
a€ected fruits showed no signi®cant di€erences in wax morphology and cuticular thickness or permeability. Our
results suggest that an excessive loss of water from hypodermal and albedo cells is responsible for the disorder.
# 2001 Annals of Botany Company
Key words: Citrus, physiological disorder, cell collapse, rind blemish, fruit quality.
I N T RO D U C T I O N
`Navelate' sweet orange originated as a bud mutation on a
`Washington' navel tree in Vinaros (CastelloÂn), Spain.
`Navelate' has several advantages for Spanish citriculture,
including high fruit quality and late maturation. However,
its culture under di€erent conditions has revealed the fruit
to be highly sensitive to rind breakdown causing rind stain
during ripening (Zaragoza and Alonso, 1975). Rind breakdown has also been observed during post-harvest storage
(AlfeÂrez and ZacarõÂ as, 2001). This peel disorder causes
considerable reduction in external fruit quality and is
responsible for large reductions in the amount of exportable
fresh fruit. Up to 80 % of fruit was found to be a€ected in
the more sensitive orchards (Almela et al., 2000). `Navelina', `Washington navel' and `Lane late' are among the
navel cultivars also liable to su€er the disorder, at least
under Mediterranean climatic conditions. A similar disorder has also been described in grapefruit, `Fallglo'
tangerine (Petracek et al., 1995, 1998) and `Shamouti'
orange (Ben-Yehoshua et al., 2001).
A€ected fruits are characterized by sunken colourless
areas of the peel (Fig. 1A) which develop into reddishbrown, dry areas partially covering the exposed portion of
the mature fruits (Fig. 1B). The incidence of this disorder
varies from year to year, among groves and even among
fruits of a given tree (Zaragoza and Alonso, 1975; Almela
et al., 2000), although this has never been formally
* For correspondence. Fax ‡34 96 387 73 93, e-mail magusti@prv.
upv.es
0305-7364/01/090415+08 $35.00/00
evaluated. The period of natural occurrence of rind stain
in `Navelate' extends from the onset of the process of fruit
colour break and for several weeks thereafter, depending on
climatic conditions. Postharvest incidence of the disorder in
mature fruit at non-chilling temperatures takes place
irrespective of the harvest time, but increases quantitatively
with fruit maturity (AlfeÂrez and ZacarõÂ as, 2001). The causal
factors and their mechanism of action are not known,
although a number of suggestions have been made.
Nutritional imbalance (Chapman, 1958; Grierson, 1965),
climatic changes, such as rainy periods followed by wind
(Zaragoza and Alonso, 1975) or cold wind (Klotz et al.,
1966), sudden changes of relative humidity (AgustõÂ and
Zaragoza, 2000) and the development of di€erential water
stress across the rind of the fruit (ZacarõÂ as et al., 2001) are
involved in the development of `Navelate' rind breakdown.
Consequently, daily variations in relative humidity (RH)
and evapotranspiration in relation to the natural occurrence
of rind staining were studied.
This paper examines the rind structure and morphology
of rind-stained fruits of `Navelate' sweet orange in
comparison with healthy fruits, using light and scanning
electron microscopy. Cryo®xation of peel using liquid
nitrogen slush was carried out to prevent preparative
damage (Echlin and Burgess, 1977). Histological studies
are particularly e€ective when una€ected fruits are available as controls and allow studies of development disorders
and determination of the timing of initiation, structure and
cause of the stain (Freeman, 1976). Water permeability of
isolated cuticles was studied and epicuticular wax
# 2001 Annals of Botany Company
416
AgustõÂ et al.ÐLoss of Water and Rind Breakdown of Citrus
F I G . 1. Rind breakdown of `Navelate' sweet orange. A, First symptoms are characterized by depressed colourless areas; B, fruits with severe
symptoms of rind breakdown develop reddish-brown and dry areas. Photographs taken on 10 January (A) and 14 March (B).
morphology was also examined in a€ected fruits, since this
layer of the fruit a€ects the rate of water exchange through
the rind.
M AT E R I A L S A N D M E T H O D S
Plant material
`Navelate' sweet oranges [Citrus sinensis (L.) Osbeck] were
collected from two commercial groves (I and II) of 25- and
30-year-old trees grafted onto citrange Carrizo [Citrus
sinensis (L.) Osb. x Poncirus trifoliata (L.) Raf.], planted
6 6 m and 5 6 m apart, respectively, grown in a
medium-heavy soil with furrow irrigation and located in
CastelloÂn province, Spain. The study was conducted for ®ve
consecutive years (1995±1996 to 1999±2000). Prior to
colour break, four-tree plots and four replications per
grove were randomly selected. Each tree was divided into
four sectors: inside and outside canopy and northwest
(NW) and southeast (SE). `Outside' fruits were those found
0±0.25 m into the tree canopy. Twenty fruits from each
sector were individually rated for the presence or absence of
rind breakdown. The percentage of a€ected fruits was
analysed after arc-sin transformation and used for analysis
of variance, using the Keul's multiple range test for means
separation.
To study rind structure, fruits were collected from the
NW quadrant, at approx. monthly intervals, from colour
break (early December) to 4 months later. For each
sampling period, ®ve oranges were examined by light and
scanning electron microscopy.
Light and electron microscopy
For light microscopy, rind pieces from the exposed face of
the fruit were vacuum-in®ltrated for 10 min in 3 % glutaraldehyde in 50 mM SoÈrensen phosphate bu€er ( pH 7.2) and
®xed in the same solution for 2 h at room temperature. The
material was then rinsed several times with the same bu€er,
AgustõÂ et al.ÐLoss of Water and Rind Breakdown of Citrus
dehydrated in a graded ethanol series for 4 h, and embedded
in LR white. Polymerization was at 60 8C for 24 h. Sections
(about 1mm thick) were cut with a Reichert Ultracut
ultramicrotome using glass knives and ®xed to microscope
slides. Sections were stained with 1 % aniline blue-black in
7 % acetic acid for general proteins (Fischer, 1968), followed
by counterstaining with periodic acid and Schi€'s reagent
for insoluble polysaccharides (Feder and O'Brien, 1968),
and with a saturated solution of sudan IV in 70 % ethanol
for lipids, cuticle, suberized walls and tannins (O'Brien and
McCully, 1981). Bright-®eld micrographs were taken with a
Leitz-Orthophlan microscope.
To examine the rind epidermis and epicuticular wax by
scanning electron microscopy (SEM), rind pieces were ®xed
and dehydrated as above. The material was critical point
dried in liquid carbon dioxide and mounted on SEM stubs.
The mounted material was coated with gold in a Polaron E6100 sputter coater. SEM was performed on an ISI-DS 130
scanning electron microscope at an acceleration voltage of
10±15 kV. To examine the cuticle and ¯avedo and albedo
tissues by cryo-SEM, small blocks (approx. 0.125 cm3) of
rind were excised with a razor blade, secured in copper
stubs with water, immediately frozen in nitrogen slush
(ÿ210 8C) and mounted on SEM stubs. The mounted
material was cryo-fractured with a microtome blade cooled
to ÿ170 8C, coated with a ®lm of gold and observed on a
cold stage. SEM was performed using a JEOL JSM-5410
scanning electron microscope.
417
T A B L E 1. Proportion of `Navelate' rind breakdown-a€ected
fruit as in¯uenced by canopy position
Grove I
Grove II
Outer canopy
Exposed face
Unexposed face
Both faces
59.1 + 4.5
3.3 + 1.0
13.8 + 6.3
48.8 + 7.7
4.2 + 1.1
4.4 + 1.3
Inner canopy
Exposed face
Unexposed face
Both faces
25.7 + 5.0
6.0 + 2.0
3.4 + 1.8
9.6 + 1.3
5.5 + 0.5
0.4 + 0.1
Mean + s.e.
Values expressed as % of a€ected fruits.
Date of evaluation: 31 Jan. 1996.
station of the National Weather Service, CastelloÂn, Spain,
from 1±31 December. Daily average data are reported in
this study.
R E S U LT S A N D D I S C U S S I O N
Assessment of trees for the incidence of rind breakdown
Fruits from the two groves were sampled at maturity
from the NW quadrant. Twenty-®ve discs of rind
(2.83 cm2) were punched from the exposed portion of the
fruits. Discs from healthy portions of healthy and a€ected
fruits and discs from damaged areas of a€ected fruits were
used. Epicuticular lipids were removed by dipping discs in
chloroform for 60 s at 45 8C. Cuticles were obtained from
dewaxed discs according to Roberts et al. (1959). Detached
cuticles were carefully rinsed in deionized water until the
adhering cellular debris was completely removed, washed
for 24 h in large volumes of 2 % aqueous borax solutions
and rinsed again in deionized water.
Water permeability of the isolated cuticles was determined gravimetrically using Petri dishes of 5 cm diameter
covered with 5.5 cm diameter circles of polytetra¯uoroethylene (Te¯on) with four holes, each 0.282 cm2. Cuticles
were mounted on the holes with their inner surfaces facing
the Te¯on and sealed with silicone grease. Cuticular
damage during manipulation was assessed using binocular
lenses. Cuticles were then dried at room temperature.
Afterwards, dishes were ®lled with 5 ml deionized water
and kept at 25 + 1 8C. Dishes were periodically weighed
until 0.2 % of the initial weight was lost. For each sample,
three replications of 16 cuticles were used.
The incidence of rind breakdown of `Navelate' oranges was
examined over 5 consecutive years in two orchards. The
proportion of a€ected fruits varied from year to year and
between the two orchards. In grove I, the incidence of the
disorder varied from 28.9 + 3.3 % in 1996±1997 to
82.0 + 3.5 % in 1999±2000. In the same year (1995±1996),
the percentage of a€ected fruit in grove I was 55.1 + 3.8
while that in grove II was 36.5 + 2.7. These results illustrate
the randomness and variable incidence of the disorder and
indicate that variations in climatic conditions during the
growing season and within each particular orchard may
in¯uence the susceptibility of fruit to this peel disorder.
Rind breakdown was found in fruits located in all sectors
of the tree, but data from all 5 years consistently showed that
fruits exposed to the NW face of the tree were more prone to
develop the disorder. Table 1 shows the results for 1995±
1996 in the two groves studied. The highest proportion of
`Navelate' fruits with symptoms of rind breakdown for both
groves was found in the outer portion of the canopy. Moreover, the disorder was also more prevalent at the exposed
face of the fruit, irrespective of its location on the tree.
The in¯uence of environment on the development of
physiological fruit disorders is well established for citrus
(Agustõ , 1999). Its e€ect has been related to fruit position on
the tree (Arpaia et al., 1991) and determines the incidence of
several important disorders such as creasing (Jones et al.,
1967), splitting (Almela et al., 1994), peel pitting of
`Fortune' mandarin (Almela et al., 1992), cold pitting of
grapefruit (Cohen et al., 1994) and rind stain of `Valencia'
(Arpaia et al., 1991) and navel orange (Eaks, 1964).
Climate data
Climatic conditions
Temperature, RH, water evapotranspiration and wind
speed and direction data were collected at the weather
In general, rind breakdown appears throughout autumn
and winter, but it sometimes appears suddenly. In these
Isolation of cuticles and determination of water permeability
418
AgustõÂ et al.ÐLoss of Water and Rind Breakdown of Citrus
25
Temperature (°C)
20
recent years (Reitz and Embleton, 1986; Arpaia et al.,
1991; Syvertsen and Lloyd, 1994; Cutuli and Salermo, 1998;
AgustõÂ , 1999).
A
15
Development of rind structure of a€ected fruit
10
5
0
–5
26/11
06/12
16/12
26/12
05/01
Date
10
B
80
8
60
6
40
4
20
2
0
26/11
06/12
16/12
26/12
ETo (mm d–1)
RH (%)
100
0
05/01
Date
F I G . 2. Average daily maximum (W) and minimum (Q) temperature
(A), relative humidity (W) and evapotranspiration (Q) (B) collected
at a weather station 5 km from the experimental groves. Data
corresponding to December 1995±1996.
cases, analysis of climatic conditions can shed light on the
causes of this disorder and on its timing. For instance, one
such case occurred in 1995±1996. The ®rst symptoms
appeared on 29 December, 3 d after a period of 3
consecutive days of high temperatures, with minimum
values increasing to 18.3 8C on 25 December (Fig. 2A).
During these days, RH reached its lowest value that
monthÐ59 % on the 26 DecemberÐand consequently
evapotranspiration reached its maximum at over 5.5 mm
d ÿ1 (Fig. 2B). There followed 3 d of low temperatures,
reaching 6 8C and 80 % RH, and evapotranspiration was
reduced to 1 mm d ÿ1 (27 and 29 December) (Fig. 2A and
B). These changes coincided with a windy period from 24 to
28 December. The predominant wind direction was S±SE
and the maximum wind speed (average for a 24 h period)
was 25.5 km h ÿ1 on 26 December; this wind is usually
warm and dry in the Spanish Mediterranean coast, thus
contributing to the increased evapotranspiration. A similar
pattern of environmental conditions vs. appearance of the
disorder was observed in the following seasons (data not
shown). Thus we conclude that initial symptoms of the
blemish coincided with days of low temperature and high
RH following a period of high temperature, low RH and
high evapotranspiration.
The e€ect of abiotic factors on fruit quality, together
with commercial considerations, has been reviewed in
In citrus, the rind consists of two tissues, the exocarp or
outer rind, named the ¯avedo, and the mesocarp or albedo
(Figs 3A and 4A). Epidermal cells in the ¯avedo are
polygonal and isodiametric in shape with no intercellular
spaces. The hypodermal parenchymatous cells are located
immediately below the epidermis and surrounding the oil
glands (Fig. 3A and B). These cells are spherical to slightly
oval in pro®le, thin walled, highly vacuolated and they
increase gradually in size deeper in the rind with small
intercellular spaces. The albedo is white in colour and
consists of meristematic cells, irregular in shape and size
and with large air spaces (Figs 3A, 4A and 5A).
A structural comparison of cross-sections of the rind
from healthy and damaged fruits (Fig. 3) increased our
understanding of the morphological characteristics of
`Navelate' rind breakdown and its probable origin. Initial
symptoms of the disorder appeared in the transitional zone
of the albedo-¯avedo (Fig. 3B); the epidermal cells, the
outer layers of the ¯avedo and the deeper layers of the
albedo appeared intact for several weeks with no sign of
necrosis or collapse. Damaged tissues showed structural
alterations in a large number of subepidermal cells,
sometimes underlying oil glands that remained intact, at
least in the initial stages of the disorder. The cytoplasm of
these cells was disorganized and appeared as a mass of
collapsed material located centrally in the cell; it showed a
highly positive reaction for proteins when stained with
aniline blue-black (Fig. 3C). This intense staining is
characteristic of the later stages of degeneration processes
(Vercher et al., 1994). Functioning of the plasmalemma and
tonoplast is impaired in the collapsed cells and this may
lead to a loss of both cellular osmoregulatory capacity and
of cellular liquids in the a€ected areas; in the absence of
turgor the walls of damaged cells could not withstand
collapse (Shomer and Erner, 1989).
SEM of a€ected tissue sampled at di€erent dates showed
more accurately that `Navelate' rind breakdown begins in
the deeper layers of ¯avedo cells and the external layers of
albedo cells (Fig. 4B). A€ected cells seemed devoid of
contents and had twisted and squashed walls, forming a
layer of collapsed cells between healthy intact cells of the
¯avedo and albedo. The disorder progressed across the
outer albedo and the inner layers of ¯avedo (Fig. 4C),
reaching the epidermis in the later stages (Fig. 3D). The
cytoplasmic degradation, tonoplast disruption and the
concomitant enzymatic oxidation of the vacuolar contents,
rich in phenolic substances (Matile, 1984), might be related
to the dark-brown coloured areas characteristic of the later
stage of development of `Navelate' rind breakdown
(Figs 1B and 3D).
There were marked di€erences in the structure of the
albedo in samples taken from healthy areas or from
breakdown-a€ected areas of the rind (Fig. 5A and B). In
healthy fruits, albedo cells were deeply lobed and, at the
AgustõÂ et al.ÐLoss of Water and Rind Breakdown of Citrus
419
F I G . 3. Cross-sections of rind from the north-western exposed face of a `Navelate' sweet orange. Healthy fruits (A) and fruits with initial (B and
C) and well developed (D) symptoms of rind breakdown. Sections were stained with aniline blue-black/periodic acid-Schi€'s reagent and Sudan
IV. B, A group of collapsed cells located in the outer layers of the albedo and the deeper layers of the ¯avedo (arrows) stained strongly. C, Flavedo
and albedo cells showing signs of cytoplasm breakdown (arrows). D, Epidermal and hypodermal cells are strongly stained and crushed; the
surface of the fruit is sunken. Og, Oil gland; Fl, ¯avedo; Al, albedo.
connections between cells, the walls extended to form
tubular arms. The intercellular air spaces, large in size, are
responsible for the spongy texture of albedo tissue at fruit
maturity (Fig. 5A), as reported by Storey and Treeby (1994)
for `Leng' navel and `Valencia' sweet oranges. In damaged
fruits, albedo cells seemed to lack content and were
¯attened, with the walls appressed (Fig. 5B). In contrast
to creasing (Storey and Treeby, 1994), albedo cells were not
separated at the middle lamella, forming a mass of tissue
with very large irregular air spaces and showing irreversible
damage and loss of turgor which provoked cell breakdown.
The micrographs suggest that loss of cell turgor might be
the cause of the disorder rather than cell separation or
fracture of the cell walls.
Epicuticular wax morphology and cuticular permeability
An excessive loss of water might be responsible for the
crushed appearance observed in ¯avedo and albedo cells.
To test this possibility, the structure of the cuticle was
studied by SEM and its water permeability determined.
The barrier against di€usion of water through the cuticle
depends on the amount, composition and structure of
cuticular waxes (SchoÈnherr, 1982; Geyer and SchoÈnherr,
1990). Surface wax morphology of healthy and damaged
areas of `Navelate' fruits with early symptoms of rind
breakdown were similar (Fig. 6A and B). In both cases, the
wax layer on the surface was largely amorphous showing a
high density of small plate-like structures. The surface of
damaged areas of a€ected fruits was rough and had an
undulating and depressed appearance (Fig. 6B), resembling
that shown in cross-section (Fig. 3B and D); this is to be
expected since damaged areas are characteristically sunken
(Fig. 1). On the other hand, the cuticle of damaged areas of
a€ected fruit was morphologically normal and showed no
signs of disruption in comparison with that of healthy fruit
(Fig. 7A and B). Cuticle thickness remained constant
regardless of the incidence of the disorder.
Water permeability of isolated cuticles from healthy and
a€ected fruits was similar for both sampling dates
(Table 2). Moreover, there were no signi®cant di€erences
between healthy areas and damaged areas of the same
a€ected fruit. This is because the epidermis of a€ected fruits
remains intact until the later stages of rind breakdown and
there were no di€erences in rind wax morphology. This
rules out temperature as the direct causal factor since, if this
420
AgustõÂ et al.ÐLoss of Water and Rind Breakdown of Citrus
F I G . 5. Cryo-SEM of albedo from healthy (A) and damaged mature
fruits (B) of `Navelate' sweet orange. The disorder does not separate
cell walls at the middle lamella and does not fracture cell walls.
Damaged albedo typically shows ¯attened cells and very large and
irregular intercellular air spaces (E).
T A B L E 2. Water permeability (mg cm ÿ2 h ÿ1) of isolated
cuticles from healthy and rind breakdown-a€ected fruits of
`Navelate' sweet orange
Rind breakdown-a€ected fruit
A€ected area
Healthy area
Healthy fruit
F I G . 4. Evolution of `Navelate' rind breakdown shown by cryo-SEM.
A, Cross-section of rind from healthy fruit. B, Early stages of rind
breakdown; deeper cells of the ¯avedo seem devoid of content and are
¯attened (arrows). C, Terminal stages of rind breakdown showing very
large intercellular air spaces; advance of the collapse both towards the
inside (albedo) and outside of the fruit reaching the epidermis. E,
Epidermis; Fl, ¯avedo; Al, albedo.
was the case, the epidermis should be the ®rst tissue to be
a€ected, as has been reported for citrus (Pantastico et al.,
1968; Chalutz et al., 1985; Vercher et al., 1994), and the
pattern of wax secretion from the epidermal cells should be
8 Feb. 1996
14 Mar. 1996
10.4 + 1.1
nd
10.3 + 0.8
11.3 + 0.7
11.0 + 0.7
10.4 + 0.5
Mean + s.e.
nd, Not determined.
modi®ed (Geyer and ShoÈnherr, 1990). Therefore, we may
conclude that rind breakdown in `Navelate' oranges
provokes collapse of subepidermal cells without a€ecting
the morphology and water permeability of the cuticle.
In conclusion, sudden changes in relative humidity at
fruit colour break seem to be responsible for the natural
development of rind breakdown of `Navelate' oranges. This
is in agreement with observations under postharvest
AgustõÂ et al.ÐLoss of Water and Rind Breakdown of Citrus
421
F I G . 7. Cross-sections through cuticle (arrows) and epidermis from
healthy (A) and damaged areas (B) of the same fruit. Photograph taken
on 8 February.
F I G . 6. Epicuticular wax morphology of healthy (A) and damaged (B)
areas from the north-western exposed face of `Navelate' sweet orange.
In both cases, the surface shows a high density of small wax platelets
embedded in an amorphous wax layer. Photograph taken on 14 March.
conditions in which rind breakdown was aggravated by
transferring fruits at 20 8C from 45 to 95 % RH (AlfeÂrez
and ZacarõÂ as, 2001). Some hypodermal cells and neighbouring albedo cells lost water, membranes were damaged
and cells collapsed; afterwards, tissues of the a€ected area
die, sink and form slowly developing reddish-brown
necrotic lesions. The reason for this loss of water is unclear
since the rind epidermis remains intact for several weeks
following the onset of the disorder and there are no
di€erences in rind wax morphology and cuticular permeability between healthy and damaged fruits.
AC K N OW L E D GE M E N T S
This research was supported by grants from Generalitat
Valenciana, ConsellerõÂ a d'Agricultura, Pesca i AlimentacioÂ
(Projecte GV-CAPA 97-01). We thank Mr M. Planes
(Electron Microscopy Service, Universitat PoliteÂcnica,
Valencia) and Mr V. Segura for technical advice and
assistance.
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