Scientia Horticulturae 81 (1999) 279±286

Microstructure of the skin of d'Agen plums

R. Storeya,*, W.E. Priceb
a
CSIRO Plant Industry ± Horticulture Unit, Merbein Laboratory, P.M.B.,
Merbein, Victoria 3505, Australia
b
Department of Chemistry, University of Wollongong, Northfields Avenue,
Wollongong, New South Wales 2522, Australia

Accepted 7 January 1999

Abstract

The skin of mature d'Agen plums (Prunus domestica L.) was investigated by cryo-scanning
electron microscopy. Regions of the surface of the fruit were differentiated by the presence or
otherwise of a waxy bloom. The epicuticular wax consisted of an underlying amorphous wax layer
adjacent to the cuticle proper together with crystalline granules of wax protruding from the surface.
Over non-bloom regions of the skin the number and size of the granules were small and the
underlying amorphous wax layer predominated. Differences between bloom and non-bloom regions
of the skin were attributed to variations in the microclimate of fruits, such as incident radiation. The
cuticular membrane was >5 mm thick. The cuticular layer showed layering, but the lamellation of
the cuticular layer, as observed by SEM, did not correspond to any recognised cuticle structural
types based on light microscopy and transmission electron microscopy of plant cuticles. Fractures
of the epicuticular wax layer frequently formed along the long axis of the stomatal guard cell pair.
Localised water loss and collapse of dermal cells occurred in the area around stomata with large
fractures. # 1999 Elsevier Science B.V. All rights reserved.

Keywords: d'Agen plum; Skin morphology; Epicuticular wax; SEM; Drying

1. Introduction

The cuticle (cuticular membrane) of plants is a noncellular, nonliving, lipoidal

membrane (Bukovac et al., 1981) and forms a major barrier to the movement of
water and solutes into and out of plants (SchoÈnherr and Riederer, 1989). The

* Corresponding author. Fax: +61-3-5051-3111; e-mail: richard.storey@pi.csiro.au

0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 2 4 - 2
280 R. Storey, W.E. Price / Scientia Horticulturae 81 (1999) 279±286

cuticle is composed of the biopolymer, cutin, with embedded intracuticular

waxes; further waxes may be deposited on the surface of the cuticle as an
epicuticular wax layer (Chamel, 1986). The structure of plant cuticles is not
homogeneous and a number of models have been proposed by Holloway (1982).
The epicuticular wax may form a partial or continuous layer of amorphous wax
over the surface of higher plants (Baker, 1982), and, in many fruits, crystalline
wax structures are extruded to the external surface giving fruits their
characteristic waxy bloom (Jeffree et al., 1975).
The microstructure of the epicuticular waxes of plums was first studied by
several researchers in the 1960s (Skene, 1963; Bain and McBean, 1967, 1969). In
this era, researchers used carbon replica (Juniper and Bradley, 1958) and double
stage plastic replica (Mueller et al., 1954) methods to study the structure of plant
surfaces. Both methods may damage surface waxes and produce artefacts. The
plastic replica method `wets' the plant surface (Juniper and Bradley, 1958) while
the carbon replica method may radiate heat, from the high-temperature carbon
arc, on to the surface of the specimen (Schieferstein and Loomis, 1959). Leece
(1978) reported on the ultrastructure of the surface waxes of leaves of Prunus
domestica L. using both freeze dried and air dried plant material. Bain and
McBean (1969) prepared thin sections of the plum skin after stabilizing the
surface waxes by evaporating metal on to the fruit surface. Changes to the
microstructure of epicuticular waxes through the use of organic solvents during
conventional methods of preparation of plant material for electron microscopy
have been reviewed comprehensively by Reed (1982).
The development of cryo-scanning electron microscopy has overcome the
detrimental effects of many of these steps in the preparation of plant material. During
sputter coating the temperature of the specimen is held at ÿ1208C so that little
damage is likely to occur to the crystalline structure of the epicuticular wax. There
have been only a few applications of cryo-scanning electron microscopy to the study
of the surface waxes of Prunus spp., (e.g. leaves, Fuchigami et al., 1981). P.
domestica L. cv. d'Agen is the major plum variety used in Australia to produce
prunes. In this paper we report on a brief study of the surface and transverse sectional
structure of the skin of d'Agen plum using cryo-scanning electron microscopy. Our
results are compared to earlier, non-cryogenic studies, and variations in the
microstructure and continuity of the epicuticular wax are reported.

2. Materials and methods

2.1. Plant material

Mature d'Agen plums (P. domestica L.) were collected from a commercial
orchard near Loxton in SA, Australia. The climate of Loxton is hot and dry with
an average annual maximum temperature of 23.38C and annual rainfall of
 R. Storey, W.E. Price / Scientia Horticulturae 81 (1999) 279±286 281

275 mm. From November, 1994 to March, 1995 average monthly daily maxima
were 26, 32, 32, 31 and 268C. Over this period, the daily maximum equalled or
was greater than 358C on 31 days with 9 days reaching a top temperature of 398C.
Fruit were picked at the beginning and end of March. The plums were transported
in polystyrene containers packed with shredded paper to prevent damage to the
microstructure of the epicuticular wax. The plums were stored for several weeks
at 48C without deterioration in the condition of the fruit as judged by declining
fruit turgor and the absence of fungal hyphae.

2.2. Cryo-scanning electron microscopy

To examine the microstructure of the plum surface, thin tangential slices of skin
were excised with a new razor blade and positioned on an aluminium stub
covered in conductive carbon cement. The specimen was frozen rapidly by
plunging the stub into liquid nitrogen (ÿ1968C). The stub was transferred under
liquid nitrogen to a Balzers SCU 102 preparation chamber which was attached to
a Philips 500 scanning electron microscope (SEM). A Yamatake-Honeywell DCP
500 digital controller regulated the temperature of the cold stage in the
preparation chamber and the microscope. The stub was etched by warming to ca.
ÿ968C then re-cooled to ÿ1308C to remove any ice crystals formed on the
surface of the specimen during the transfer from the Dewar flask to the
preparation chamber. The specimen was sputter coated with a thin layer of
conductive gold and transferred to the microscope chamber.
To obtain transverse freeze fractures of the skin, thin strips of skin were excised
and inserted into the round slot of an aluminium stub containing a small volume
of water. The stub was frozen in nitrogen slush (ÿ2108C) and transferred to the
Balzers preparation chamber under liquid nitrogen. The specimen, maintained at
ÿ1308C, was fractured with the Balzers microtome, which was set up with a
Feather microtome blade cooled to ÿ1508C. The specimen was etched to enhance
definition of cells and cell structure, and then coated with gold as described
above.
The SEM was operated at 12 kV and the cold stage maintained at a temperature
of ÿ1408C. Grid voltage of the detector was varied to capture both secondary and
backscattered electrons. Backscattered electron micrographs were used to
highlight specimen topography. More than 15 fruit were examined in the SEM
and representative micrographs of our observations are presented.

3. Results and discussion

The epicuticular wax of mature d'Agen plum taken from regions of the skin
showing a waxy bloom consisted of an outer crystalline layer with an underlying
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amorphous layer (Fig. 1(A)). Skene (1963) described the crystalline structure of
the surface waxes of Ontario plums as fibrils and small thin platelets. Some of the
fibrils appeared hollow although the author conceded that this might have been an
artefact arising during sample preparation. Bain and McBean (1967, 1969)
described the crystalline microstructure of the epicuticular wax of mature d'Agen
plum as an underlying wax layer of platelets with protuberances some of which
were tubular. In these studies replica methods of sample preparation were
employed with their associated inherent drawbacks as described previously. Our
studies using cryo-preservation techniques showed that the epicuticular wax of
d'Agen plum was a closely packed granular structure overlying a more
amorphous layer. We found no evidence of hollow rods or protuberances.
We did observe major differences in the crystalline form of the epicuticular
wax on the bloom and non-bloom side of the fruit (Fig. 1(A,B)). The finer
granularity and lower density of the crystalline granules on the non-bloom side of
the fruit are consistent with less scattering of incident light and, therefore, a
reduced waxy bloom. The formation of the bloom over the surface of fruits is
probably influenced by the microclimate of the fruit, such as the effect of incident
radiation on skin temperature. Ambient temperatures in the Riverland of SA
during the summer can reach ca. 408C on some days and the temperature of the
sun-exposed surface of the plum skin may, therefore, be more than 508C (Mrozek
and Burkhardt, 1973). The epicuticular wax of d'Agen plum undergoes a rapid
(<2 h) phase transition from a crystalline to amorphous structure at a temperature
between 55 and 608C (unpubl. data). Differences in the fine crystalline structure
of the epicuticular wax of sun-exposed and shaded sides of citrus and apple fruits
have been reported (Knuth and Stosser, 1987; El-Otmani et al., 1989; McDonald
et al., 1993; Storey and Treeby, 1994). It is well established that environmental
factors such as temperature, light and humidity may modify the composition and
fine crystalline structure of plant epicuticular waxes (Riederer and MarkstaÈdter,
1996 and references therein). Thus, the variation in microstructure of the wax
between the bloom and non-bloom regions of the fruit is probably the result of
localised differences in environment, particularly variations in incident radiation
within the canopy of the tree.
Stomata, lenticels and superficial fractures of the cuticle modulate the
permeance of cuticular membranes (Glenn and Poovaiah, 1989). Closed stomata
may have conductances of the same magnitude or greater than the permeance of
the cuticular membrane (Kerstiens, 1996). However, some of the stomata of
d'Agen plum were totally occluded with extruded wax (Fig. 1(C)) which is likely
to reduce their conductance. Stomata covered in wax as shown in Fig. 1(C) are
probably dysfunctional. The epicuticular wax of fruits, such as, apple (Knuth and
Stosser, 1987), pear (KovaÂcs et al., 1994), nectarines (Nguyen-The, 1991) and
plum (Mrozek and Burkhardt, 1973) frequently fracture during fruit growth. We
observed very few cuticular fractures over the surface of d'Agen plums apart
 R. Storey, W.E. Price / Scientia Horticulturae 81 (1999) 279±286 283

Fig. 1. Surface views of the epicuticular wax layer of mature d'Agen plums. Crystalline granules
on the bloom side of the fruit (A, bar ˆ 2.5 mm). Predominately amorphous epicuticular wax with
smaller and fewer crystalline granules on the non-bloom side of the fruit (B, bar ˆ 2.5 mm).
Crystalline layer of epicuticular wax covering a stomate (C, bar ˆ 5 mm). Fracture along the long
axis of an elliptical stomate (D, bar ˆ 20 mm).

from those associated with stomata (Fig. 1(D)). Our observations concur with
those of Mrozek and Burkhardt (1973) that stomata act as nucleating sites for the
formation of fractures in the cuticular membrane. The depths of the fractures
appeared to be relatively shallow, confined mainly to the epicuticular wax layer.
Stomata of fruits, such as grapes (CommeÂnil et al., 1997) and plums (Mrozek and
Burkhardt, 1973) may degenerate and form lenticels which may further modify
the local permeance of the cuticle to water. Observations using a backscattered
electron signal showed that dermal layers of fruits frequently collapsed in the area
surrounding stomata with large cuticular fractures (data not shown). This was
strong evidence for the movement of water from dermal cells to the atmosphere,
leading to cell collapse, but clearly, the effect remained localised to a small region
of the fruit surface.
The skin of the d'Agen plum was made up of several dermal layers, which were
slightly tabular in shape (Fig. 2(A)). Parenchyma cells of the fruit flesh increased
284 R. Storey, W.E. Price / Scientia Horticulturae 81 (1999) 279±286

Fig. 2. Transverse freeze fractures through the skin of mature d'Agen plums. Overview of the
dermal and parenchyma cells of the outer flesh (A, bar ˆ 80 mm). Epicuticular wax, cuticular
membrane and epidermal cell of the skin from the bloom side of the fruit (B, bar ˆ 5 mm). ICL-
internal cuticular layer, ECL-external cuticular layer, CP-cuticle proper, CW-cell wall, CY-
cytoplasm, DL-dermal layers, EW-epicuticular wax, P-pulp, V-vacuole.

progressively in size with distance from the epidermis. Intercellular air spaces of
the parenchyma cells were small. A transverse fracture through the skin on the
bloom side of the fruit showed that the epicuticular wax layer (ca. 5 mm) was
made up of an outer crystalline and an inner amorphous layer (Fig. 2(B)). The
epicuticular wax showed a marked discontinuity between it and the cuticle
proper. Bain and McBean (1969), using conventional methods for the preparation
of transverse sections, were able to differentiate an epicuticular wax layer, a
cuticle and cell wall. Freeze fractures of the skin (Fig. 2(B)) showed that the
cuticular membrane of d'Agen plums, in particular the cuticular layer, was
structurally heterogeneous. It was possible to differentiate four to five discrete
layers which made up the cell wall, cuticular layer and cuticle proper (Fig. 2(B)).
The demarcation between the epicuticular wax layer and the cuticle proper was
clearly evident from our observations probably because it represents a marked
change in the composition of the cuticular membrane. The cuticle proper is
composed of cutin, a polyester of hydroxylated fatty acids with embedded waxes,
whereas the epicuticular wax layer consists, predominantly, of soluble fatty acids
(Baker, 1982; Chamel, 1986). The sublayers of the cuticular layer in Fig. 2(B)
might represent both chemical and/or temporal transitions within the cuticular
membrane. It was not, however, possible to correlate the horizontal lamellation of
the cuticular layer to models of cuticle structure as proposed by Holloway (1982).
 R. Storey, W.E. Price / Scientia Horticulturae 81 (1999) 279±286 285

The structural types described by Holloway were based on observations using

light microscopy and transmission electron microscopy (TEM) techniques. Thus,
the multiple lamellation of the cuticular layer, as observed by cryo-SEM, may not
necessarily correspond to the reticulate and lamellate layers of the cuticular layer
when observed by TEM.

Acknowledgements

The authors wish to thank Mr W. Berrens of Loxton, SA, for supplying the
plant material. The research was partially funded by a grant from Dried Fruits
Research and Development Council (DFRDC), the receipt of which is much
appreciated. The authors acknowledge the comments by Dr. Chris Jeffree on
matters of interpretation of micrographs.

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