Download Structure and Nanostructure of the Outer Tangential Epidermal Cell

Structure and Nanostructure of the Outer Tangential Epidermal
Cell Wall in Vaccinium corymbosum L. (Blueberry) Fruits by
Blanching, Freezing–Thawing and Ultrasound
J. Fava,1 S.M. Alzamora2,3 and M.A. Castro1,*
1
2
Laboratorio de Anatomía Vegetal, Departamento de Biodiversidad y Biología Experimental
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad
Universitaria, C1428 EHA, Buenos Aires, Argentina
3
Consejo Nacional de Investigaciones Científicas y Técnicas
The design of minimal technologies for blueberries preservation requires, among others, the knowledge
of structural and ultrastructural cell changes during the processing. This work examined the main structural alterations that occurred in the outer tangential epidermal cell wall of fruits of Vaccinium corymbosum L. (blueberries) due to blanching, freezing–thawing and ultrasound. Light microscope (LM),
environmental scanning electron microscope (ESEM), transmission electron microscope (TEM) and
atomic force microscope (AFM) observations were analysed and discussed. Each treatment produced
specific effects on the outer tangential epidermal cell wall of the epicarp: swelling and rupture of the
inner and outer tangential cell wall by blanching; and cell wall shrinkage and rupture by ultrasound; and
folding and compression of the epicarp by freezing–thawing. After treatments, a delimited transition
between the cuticle, the cutinised layer and the cellulosic layer on the outer tangential epidermal cell
wall was observed in all treated fruits.
Key Words: blueberry, nanostructure, cell wall, freezing–thawing, blanching, ultrasound
INTRODUCTION
Blueberries have become increasingly popular
because of their health-promoting properties (mainly
antioxidant ones). High quality fruits with fresh-like
attributes are now demanded by consumers, to satisfy
those market requirements, the safety and quality of
fruits must be based on substantial improvements in
traditional preservation methods or on the use of
emerging technologies. Whole blueberries are usually
preserved by freezing or by controlled atmosphere
storage. However, CO2 enrichment of storage atmosphere delayed fruit senescence but may cause softening of the berries because of structural changes
produced (Allan-Wojtas et al., 2001). In the same way,
leakage of pigmented exudates through the skin cracks
and ruptures of frozen blueberries during thawing and
*To whom correspondence should be sent
(e-mail: [email protected]).
Received 11 May 2005; revised 17 November 2005.
Food Sci Tech Int
2006; 12(3):241–251
© 2006 SAGE Publications
ISSN: 1082-0132
DOI: 10.1177/1082013206065702
firmness loss may detract from product appearance
(Sapers et al., 1985). So, the design and optimisation of
minimal processes for blueberries preservation
requires, among others, the knowledge of structural
and ultrastructural cell changes due to treatments.
Dynamic changes in the chemical composition as
well as in the tissue structure during ripening, senescence, storage, post-harvest and processing of fruits
cause variation in sensory, chemical and physical properties (Kunzek et al., 1999).
In plants, the epidermal cell wall reduces the uncontrolled loss of water and apoplastic solutes; forms a
mechanical barrier against penetration by fungal
hyphae; protects tissue from mechanical damage;
reflects and attenuates radiation; acts as an accumulation compartment for lipophilic compounds, performs
a medium for plant signals and reduces water retention
on the plants surface (Kerstiens, 1996).
The epidermal cell wall can be distinguished from
all other cell walls of plants by the presence of thick
continuous lipidic layers (cutinised layer, cuticle and
epicuticular waxes) deposited on its outermost region
(Hallam, 1964; O’Brien, 1967; Hallam and Juniper,
1971; Jeffree et al., 1976). The number, thickness and
demarcation of layers varies considerably according to
species and stage of development and cannot be gener-
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J. FAVA ET AL.
alised in a cell wall structural scheme. Using fine structure as a criterion, different authors have reported six
basic morphological epidermal cell wall types (Holloway, 1982). In general, the following layers have
been described from outer to inner: (1) epicuticular
waxes (amorphous layer, crystalline or semi-crystalline); (2) cuticle (constituted only by cutin); (3)
cutinised or fibrillar layer (mainly composed by polysaccharide matrix, cutin and intracuticular waxes); (4)
pectic layer; and (5) cellulosic layer (Esau, 1953; Holloway, 1982; Baker, 1982; Lyshede, 1982; Walton, 1990;
Barthlott et al., 1998).
Ontogenetic variations in wax composition have
been mentioned in Triticum spp. (Tulloch, 1973),
Prunus persica L. (Baker et al., 1979), Citrus spp.
(Freeman et al., 1979a) and Vaccinium ashei. Rodlet
structure waxes in blueberries were due to the presence
of -diketones (Freeman et al., 1979b). Von WettsteinKnowles (1974) reported that the loss of rodlet structure waxes was coincidental with the decrease in
-diketones. This author established the contribution of
-diketones to wax tube structure in barley (Hordeum
vulgare L.). Likewise, degradation of rodlet structure
waxes are reported by Freeman et al. (1979b) in some
areas on mature fruits of V. ashei Reade (rabbiteye
blueberry). The ultrastructure of wax on ripe berries
varied from narrow, short and stubby rodlets to amorphous waxes. Chemical studies determined that -diketones constituted the major fruit wax fraction, up to
62% of the total wax (Freeman et al., 1979b).
The non cutinised cellulosic layer is a strong
network of cellulose microfibrils linked by hydrogen
bonding to xyloglucans, the non cellulosic polysaccharides. Pectic polysaccharides and additional minor cell
wall constituents (structural proteins, enzymatic proteins, hydrophobic compounds and inorganic molecules) have also been determined (Carpita and
Gibeaut, 1993; Rose and Bennett, 1999; Whitney, et al.
1999; Roberts, 2001; Cosgrove, 2001).
Minimal processing technologies based on a combination of a mild disinfection or reduction by 102 to
105 pathogenic and/or spoilage microorganisms (for
instance by blanching or by high power ultrasonic
treatment) followed by refrigerated storage or osmotic
dehydration (according to the shelf life intended) could
be an alternative to freezing and controlled atmosphere preservation methods. However, there is no
information in the literature about the structure
changes provoked in blueberries by these treatments.
The present study examined the main alterations
that occurred in the outer tangential epidermal cell
wall of V. corymbosum L. (highbush blueberry) fruits
due to blanching, ultrasound and freezing–thawing
(this last treatment was for the purpose of comparison)
using complementary microscopic techniques. These
results, along with additional in-depth inner
microstructure examination and integration with
macroscopic mechanical and sensory analysis, would
help to identify the causes of texture changes and
provide new insights to design successful minimal
preservation techniques.
MATERIALS AND METHODS
Material
V. corymbosum L. (highbush blueberry) O´Neal var.
mature fruits were collected from ‘La Kiwera’ (Mercedes, Buenos Aires province, Argentina).
Whole blueberry fruits (about 1 cm in diameter)
were subjected to freezing–thawing, ultrasound or
blanching.
Fruit Treatments
Freezing–Thawing
Blueberries were packaged in an aluminium foil.
Freezing was carried out in a domestic freezer at
20 °C for 72 h. Thawing was performed at room temperature (22–24 °C) after 2 h.
Ultrasound
Ultrasonic treatment was carried out in a 150 mL
double wall cylindrical vessel (diameter: 6.3 cm; height:
7.6 cm) connected to a thermostatically controlled water
bath (HAAKE, Model Rotovisco RV12, Germany)
whose temperature was fixed to attain 45.0 ± 0.2 °C.
Ultrasound (Vibracell®, net power output: 600 watts,
Sonic and Materials Inc., Chicago) at 20 kHz and
95.2 m (80%) of wave amplitude was applied over
5 min. The equipment had automatic amplitude compensation to ensure uniform probe amplitude regardless
of the varying loading conditions and line voltage fluctuation. The probe was previously calibrated following the
steps suggested by the manufacturer (Sonic and Materials Inc., Operator’s Manual for 600 watts Vibracell®).
Blanching
Heating was performed in saturated vapour for
2 min and then the fruits were cooled over 5 min in a
water bath at 10 °C.
Microstructural Study
Four techniques were used: environmental scanning
electron microscope (ESEM), transmission electron
microscope (TEM), light microscope (LM) and atomic
force microscope (AFM).
Rectangular strips of epicarp (having dimensions of
1.5 cm length, 1 cm width) were excised and mounted
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Structural and Ultrastructural Cell Blueberries
on metal supports. Samples were observed using a
Philips XL 30 ESEM operating at 10.0–20.0 kV and
0.8–1.5 torr.
For TEM, epicarp samples of about 1 mm3 were
excised with a scalpel and fixed in 3% (v/v) glutaraldehyde in 20 mM phosphate buffer, pH7, at 20 °C for 2 h.
Samples were washed with the buffer for 1 h and postfixed in 1% (w/v) OsO4 aqueous solution at 20 °C for
2 h. Samples were washed with the same buffer for 1 h,
dehydrated through a graded acetone series at 20 °C,
and embedded in low viscosity Spurr resin (Spurr,
1969). Ultrathin sections (1 m thick) were cut using a
glass knife with a Sorvall MT 2-B ultracut microtome,
collected on copper grids and double stained with
uranyl acetate and Reynolds lead-citrate (Reynolds,
1963). Sections were examined using a JEOL JEM
1200 EX II transmission electron microscope at an
accelerating voltage of 80 kV.
Transverse ultrathin sections (1 m thick) were
stained with toluidine blue and examined under a Zeiss
Phomi III microscope for light microscopy
(Oberkochen).
Histochemical tests for light microscopy were used
to detect cutin and pectic polysaccharides in the outer
tangential epidermal cell wall. Hand transverse sections of fresh fruits were stained with sudan IV for
cutin and ruthenium red for pectic polysaccharides
(O’Brien and McCully, 1981; D’Ambrogio de Argüeso,
1986).
For AFM, ultrathin sections were cut using a glass
knife with a Sorvall MT 2-B ultracut microtome, collected unstained on copper grids, mounted on mica
disks and examined in a Nanoscope III Atomic Force
Microscope controlled with a MultiMode Head
(Digital Instruments, Santa Barbara, CA, USA) under
dry N2 (Baker et al., 2000; Ridout et al., 2002). Images
(deflection or error signal and topography) were
acquired by using the tapping mode. The AFM tips
(silicon nitride cantilevers) were 125 m long and had a
nominal spring constant of 0.4 N/m (TAP 300, Nano
Devices). The scan area sizes varied from 10,000 to
1 m2. The scan rate applied was 1–5 Hz.
RESULTS AND DISCUSSION
Macroscopic Features
In superficial view, fresh (control) fruits, up to
10 mm in diameter, were greyish blue, firm and
rounded in shape. The presence of epicuticular waxes
determined their dull surface. The epicarp exhibited an
intact and uniform general aspect. After treatment,
processed berries showed different macroscopic features. Frozen–thawed fruits presented a less dull
surface, and an intense blue colour similar to the
control. Rounded in shape, they were very soft and did
1 cm
243
A
B
C
D
Figure 1. V. corymbosum L., control and treated
fruits, general aspects: A, mature raw fruit; B, frozen
thawed fruit; C, ultrasonicated fruit; D, vapour
blanched fruit.
not offer resistance to manual compression. Ultrasound treated fruits showed an intense dark violet dull
surface, appeared irregular in shape and did not
present resistance to manual compression, but exhibited a more consistent texture than the one of blanched
and frozen–thawed fruits. Vapour blanched fruits, with
shiny violet to black surface colour, showed a rounded
shape. After manual compression, they recovered their
original shape. Following blanching, the epicuticular
wax layer was altered and fruits became slippery to
tacky (Figure 1(A)–(D)).
Microscopic Features
Raw Fruits
The epicarp of raw fruits (control) appeared fissured
and reticulated. Deep fissures showed a sinuous trajectory and delimited smooth and reticulated intermixed
zones. In transverse sections, from outer to inner, the
outer tangential epidermal cell wall revealed the presence of epicuticular waxes, a cuticle, a cutinised layer
and a cellulose layer (Figure 2(A)–(B)).
Light microscopy observations showed the epicarp
composed by one stratum of epidermis and two or
three subepidermic collenchymatous layers. Epidermal
cells exhibited rectangular shape, parietal cytoplasm,
turgent central vacuole, thick outer and inner tangential walls, and thin radial walls. Part of the edible pulp
or mesocarp exhibited rounded to irregular cells, conspicuous intercellular spaces and turgent cells with
parietal cytoplasm. The presence of grouped lignified
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J. FAVA ET AL.
ce
c
pc
p
50 µm
A
20 µm
B
*
*
EP
M
E
100 µm
C
40 µm
D
*
F
45 µm
E
Figure 2. V. corymbosum L., raw fruit. A–B, ESEM micrographs: A, fissured and reticulated epicarp surface; B,
outer tangential epidermal cell wall (transverse section, detail). C–E, LM micrographs: C, epicarp and mesocarp
(transverse section, general aspect); D–E, transverse hand sections of the epicarp, histochemical tests: D, with
sudan IV: cutin in cuticle (white asterisk) and cutinised layer (black asterisk); E, with both sudan IV and ruthenium red: presence of pectic polysaccharides (arrow and asterisk). F, scheme of the outer tangential epidermal
cell wall (adapted from Lyshede, 1982). c cuticle; ce epicuticular waxes; E epicarp; Ep epidermis;
M mesocarp; p cellulosic layer; pc cutinized layer; waxes; cutin; pectins; — cellulose.
sclereids would contribute to increase the firmness of
mature fruits (Figure 2(C)).
Transverse hand sections treated with sudan IV
showed the occurrence of cutin in the cuticle and the
cutinised layer. The cuticle appeared intensely orange
(Figure 2(D), white asterisk). The light orange stain of
the cutinised layer indicated a lower proportion of
cutin (Figure 2(D), black asterisk).
Transverse hand sections treated with both sudan
IV and ruthenium red revealed the intensely purple
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Structural and Ultrastructural Cell Blueberries
stained pectic layer demonstrating the presence of
pectic polysaccharides. The cutinised layer was redpurple and light orange, representing the existence of
both cutin and pectic polysaccharides (Figure 2(E)
and (F)).
The TEM study of the outer tangential epidermal
cell walls showed the epicuticular waxes as a discontinuous, thin and electron-lucent stratum. The cuticle
layer appeared well defined, amorphous, smooth,
about 0.7 up to 1 m thick, and exhibiting two well
delimited zones: the electron-less dense outer zone and
the electron-dense inner zone. The limit between the
245
two last mentioned layers followed a sinuous trajectory.
In addition, an abrupt delimitation between the
cuticle and the cutinised layer was observed. The
cutinised layer (about 1.8 m thick) and the cellulosic
layer (about 1.6 m thick) appeared electron-dense
and exhibited gradual transition. The upper zone of
contact epidermal cells areas showed micropores, and
osmiophilic or electron-dense general appearance,
suggesting the presence of pectin (Figure 3(A)–(C)).
With AFM, analysed areas of 10 10 m2 and
5 5 m2 showed the outer tangential epidermal cell
c
pc
ce
c
p
pc
B
p
A
c
pc
c
pc
p
p
C
D
E
F
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Figure 3. V. corymbosum L.,
raw fruit. A–C, TEM micrographs, outer tangential epidermal cell wall: A, general
aspect; B–C, details. D–F,
AFM micrographs, transverse
sections: D, outer tangential
epidermal cell wall, general
aspect; E–F, cellulosic layer: E
general aspect; F, cellulose
microfibrils in close proximity
and parallel to the axis, detail.
c cuticle;
ce epicuticular
waxes; p cellulosic layer;
pc cutinised layer. Scales: A,
C 1 m;
B 500 nm;
D 5 5 m2; E 3 3 m2;
F 1 1 m2.
246
J. FAVA ET AL.
wall about 4–5 m thick and revealed abrupt transition
between the cuticle and the cutinised layer and gradual
transition between the cutinised and cellulosic layers.
The cutinised layer presented the highest thickness. In
areas about 3 3 m2 and 1 1 m2 the cellulosic layer
exposed cellulose fibrils, rounded to an irregular shape,
60–70 nm in diameter, in close proximity to each other
and with a distribution pattern approximately parallel
to the longitudinal axis (Figure 3(D)–(F)).
Freezing–Thawing
The epicarp showed a folded and axial wrinkled
appearance and formed longitudinal bands with a
smooth to slight furrow surface. The smooth areas
appeared between distant wrinkles. Scarce epicuticular
waxes exhibited different shapes and variable sizes
(Figure 4(A)).
Under LM, in transverse sections of frozen–thawed
fruits, the folded epicarp presented tangential
compression and scarce epicuticular materials. The
A
B
C
pc
p
C
C
D
pc
p
C
pc
p
E
F
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Figure 4. A–F, V. corymbosum L., frozen–thawed fruit.
A,
ESEM
micrographs,
epicarp surface smooth areas
and
scarce
epicuticular
waxes. B–C, LM micrographs, compressed and
folded epicarp (transverse
sections). D–F, TEM micrographs: D, thin folded radial
walls; E–F, well delimited
transition between cuticle,
cutinised layer and cellulosic
layer. c cuticle; ce epicuticular waxes; p cellulosic
layer; pc cutinised layer.
Scales:
A–C 100 m;
D 1 m; E–F 500 nm.
Structural and Ultrastructural Cell Blueberries
mesocarp appeared partially collapsed. Epidermal cells
showed disrupted membranes (tonoplast and plasmalemma), collapsed cytoplasm, thick tangential cell
walls and thin folded radial walls (Figure 4(B)–(C)).
The outer tangential epidermal cell wall, about 3.5m
thick, revealed, by TEM, the presence of a thin epicuticular wax layer separated from the cuticle. The cuticle was
amorphous, well delimited, about 0.7 to 1m thick, and
with a general aspect similar to the control. Transition
between the cuticle and the cutinised layer was well
demarcated by an electron-dense zone with a sinuous
trajectory. The cutinised layer, about 1.5–2m thick, was
electron-lucent and loose. The well-defined cutinised
layer suggested an altered distribution pattern of cutin,
waxes and polysaccharides. The electron-dense cellulosic
layer, about 0.6m thick, suggested a tight cellulose
microfibrils framework (Figure 4 (D)–(F)).
Ultrasound
The epicarp of ultrasonicated blueberries presented
a slightly folded surface with heterogeneous external
50 µm
morphology (ESEM micrographs). Smooth, warty and
grooved areas were observed. The rupture of the outer
tangential epidermal cell wall appeared like broad and
deep holes. Epicuticular waxes were partially removed
(Figure 5(A)–(B)). Under LM, epidermal and subepidermal cells showed thin and broken radial cell walls, a
thick outer tangential cell wall, and disrupted membranes (plasmalemma and tonoplast). In general, the
mesocarp presented collapsed parenchyma cells while
sclereid groups remained (Figure 5(C)–(D)).
With TEM, the epicuticular wax layers appeared
discontinuous and separated from the rest of the
epicarp. The cuticle (about 0.6–1.3 m thick) showed
electron-dense areas alternating with electron-lucent
ones. Transition between the cuticle and the cutinised
layer was delimited by a conspicuous osmiophilic or
electron-dense area which frequently protruded the
cuticle. The cutinised layer about 0.7–1 m thick was
electron-lucent. The cellulosic layer appeared well
delimited (Figures 5(E)–(G)).
With AFM, tested areas of 25 25 m2, 10 10 m2
and 5 5 m2, revealed a packed cell wall, about
50 µm
A
100 µm
C
B
100 µm
D
c
ce
pc
ce
c
p
c
pc
pc
p
p
1 µm
247
500 nm
E
500 nm
F
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G
Figure 5. A–G, V. corymbosum L., ultrasonicated fruit:
A–B, ESEM micrographs:
smooth, warty and rough
epicarp surface and holes
(arrow); C–D, LM micrographs: broken cell wall
(arrow),
disrupted
membranes and grouped sclereids;
E–G,
TEM
micrographs: electron lucent
cutinised layer and electron
dense
cellulosic
layer.
c cuticle; ce epicuticular
waxes; e sclereids; p cellulosic layer; pc cutinised
layer. Scales: A–B 50 m;
C–D 100 m;
E 1 m;
F–G 500 nm.
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J. FAVA ET AL.
p
pc
c
A
B
Figure 6. A–D, V. corymbosum
L., ultrasonicated fruit, AFM
micrographs (transverse sections): A, epidermal cell, general
aspect; B–C, outer tangential
epidermal cell wall, details;
D, cellulosic layer: cellulose
microfibrils forming a crowded
network.
ccuticle;
m
cellulose microfibrils; p cellulosic layer; pc cutinised layer.
Scales:
A 25 25 m2;
2
B 5 5 m ;
C 2 2 m2;
2
D 1 1 m .
p
pc
m
c
D
C
3.5 m thick, compacted by a mechanised effect.
Analysed areas of 2 2 m2 and 1 1 m2 showed the
cellulosic layer about 1 m thick, composed of cellulose microfibrils, rounded in shape, 30–35 nm in diameter and forming a crowded framework (Figure
6(A)–(D)).
Blanching
Vapour blanched treated fruits presented heterogeneous external morphology. Smooth, warty areas
and plates were observed. In general the surface exhibited a porous general aspect (Figure 7(A)–(B)).
In transverse sections of fruits, epidermal and
subepidermal cells revealed, by LM, the occurrence of
swollen walls. Epidermal cells presented broken inner
and outer tangential walls, occasional disrupted membranes (plasmalemma and tonoplast) and incipient
plasmolysis. The edible pulp or mesocarp apparently
remained intact (Figure 7(C)–(D)).
Heat treated blueberries, showed, by TEM, a thin
continuous epicuticular wax layer separated from the
cuticle. The cuticle, about 1 m thick, presented an
electron dense zone delimiting the abrupt transition
with the cutinised layer. The cutinised layer, about
1.5–3 m thick, appeared electron-lucent and markedly
altered by treatment, suggesting changes in the pectic
polysaccharide cell wall domain (Figure 8(A)–(C)).
With AFM, tested areas of 20 20 m2, 10 10 m2
and 5 5 m2 revealed epidermal cells with incipient
plasmolysis. The outer tangential epidermal cell walls,
about 5.5–7 m thick, showed abrupt transition
between the cuticle, the cutinised layer and the cellulosic layer. Analysed areas of 1 1 m2 showed a cellulosic layer about 4–5 m thick, with a loose cellulose
fibrils distribution pattern. Microfibrils were rounded
in shape, approximately 25–40 nm in diameter, with
inter-microfibrils spacing of about 15–30 nm. Fragments of single xyloglucans chains (about 6–8 nm in
diameter) suggested span rupture in the gaps between
cellulose microfibrils and disrupted links to one
another (Figure 8(D)–(F)).
Mechanical and thermal treatments altered the epicuticular wax layer in all treated fruits. Blanching,
freezing–thawing and ultrasound clearly emphasised
the transition between the cuticle, the cutinised layer
and the cellulosic layer; in particular, blanching distinctly modified the cutinised layer. In general, the
pectin layer was seen as a diffuse, markedly electrondense zone. Lyshede (1982) argued that, in the cell wall
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Structural and Ultrastructural Cell Blueberries
100 µm
100 µm
A
C
50 µm
100 µm
249
B
D
Figure 7. A–D, V. corymbosum L., vapour blanched fruit: A–B, ESEM micrographs: porous surface with smooth,
warty areas and plates. C–D, LM micrographs, transverse sections: broken wall (arrow), plasmolysis and occasional disrupted membranes. Scales: A, C, D 100 m; B 50 m.
TEM micrograph, the electron dense structures in the
cutinised layers and middle lamella are composed of
pectin, and this could be in accordance with the overall
staining of the wall with ruthenium red.
Frozen–thawed blueberries exhibited a compressed
and folded epicarp, cell dehydration and plasmolysis.
Probably propagation of extracellular ice caused disruption and destabilisation of membranes, and consequent loss of the capability of the plasma membrane to
act as an efficient barrier against the propagation of
extracellular ice. Yamada et al. (2002) supported the
last mentioned that irreversible changes in plant cell
wall and membranes that caused textural changes and
fruit softening.
External stress during high temperature processing
stimulated the fruits softening and reduced adhesion
between cells by pectins solubilisation. In the cellulosic
layer, vapour blanching modified the cellulose–xyloglucan network, and disturbed links to one another.
Detected xyloglucan span rupture in the gaps between
cellulose microfibrils increased the distance between
cellulose microfibrils and amplified the outer tangential
cell wall thickness.
Ultrasound caused a packed microfibril network and
the decreasing of the outer tangential epidermal cell
wall thickness. Cavitations disrupted the outer tangential epidermal cell wall and generated deep holes in the
epicarp. This alteration could contribute to the
increase in the epicarp permeability to solute uptake
during impregnation processes.
These results could help in understanding the structural responses of outer tangential epidermal cell walls
after treatments. This knowledge will improve the
design of blueberries preservation methods that
involve blanching, freezing and/or ultrasound. Likewise, the ultrastructural changes reported would be
related to texture properties and mass transport
consideration.
ACKNOWLEDGEMENTS
The present research has been supported by Buenos
Aires University (UBACyT X001), CONICET and
ANPCyT–BID. We would also like to thank Dr Sandra
Guerrero (Industry Department, FCEN-UBA) for her
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J. FAVA ET AL.
ce
c
c
pc
pc
p
p
B
A
pc
ce
p
c
c
cl
pc
p
C
D
X
m
E
F
Figure 8. A–F, V. corymbosum L., vapour blanched fruit (transverse sections): A–C, TEM micrographs: abrupt
transition between cuticle, cutinised layer and cellulosic layer, cutinised layer electron lucent; D–F, AFM micrographs, D–E, outer tangential epidermal cell wall; F, cellulosic layer, loose cellulose fibrils distribution pattern and
fragment of single xyloglucan chains. c cuticle; ce epicuticular waxes; ci cytoplasm; m cellulose microfibrils; p cellulosic layer; pc cutinised layer; x xyloglucan chains. Scales: A 2 m; B–C 1 m;
D 10 10 m2; E 5 5 m2; F 1 1 m2.
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Structural and Ultrastructural Cell Blueberries
technical assistance and to the Advance Microscopy
Centre (FCEN-UBA), SEGEMAR (INTI) and INTA
Castelar for the use of AFM, ESEM and TEM, respectively.
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