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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- Downloaded from fst.sagepub.com at UBA on June 2, 2015 242 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 Downloaded from fst.sagepub.com at UBA on June 2, 2015 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 Downloaded from fst.sagepub.com at UBA on June 2, 2015 244 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 Downloaded from fst.sagepub.com at UBA on June 2, 2015 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 Downloaded from fst.sagepub.com at UBA on June 2, 2015 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 Downloaded from fst.sagepub.com at UBA on June 2, 2015 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 Downloaded from fst.sagepub.com at UBA on June 2, 2015 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. 248 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 Downloaded from fst.sagepub.com at UBA on June 2, 2015 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 Downloaded from fst.sagepub.com at UBA on June 2, 2015 250 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. Downloaded from fst.sagepub.com at UBA on June 2, 2015 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. REFERENCES Allan-Wojtas P.M., Forney C.F., Carbyn S.E. and Nicholas K.U.K.G. (2001). 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