How Many Layers Of Cells In Tomato Skin

• Journal List
• Plant Physiol
• 5.170(two); 2016 February
• PMC4734585

Plant Physiol. 2016 Feb; 170(two): 935–946.

Ultrastructure of the Epidermal Cell Wall and Cuticle of Love apple Fruit (Solanum lycopersicum 50.) during Development^{ane, [OPEN]}

Received 2015 November 9; Accepted 2015 Dec 8.

Supplementary Materials

Supplemental Data

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Abstract

The epidermis plays a pivotal role in institute evolution and interaction with the environment. Even so, information technology is still poorly understood, especially its outer epidermal wall: a singular wall covered past a cuticle. Changes in the cuticle and cell wall structures are important to fully empathise their functions. In this piece of work, an ultrastructure and immunocytochemical approach was taken to place changes in the cuticle and the main components of the epidermal jail cell wall during tomato fruit development. A sparse and uniform procuticle was already nowadays before fruit set. During cell division, the inner side of the procuticle showed a globular construction with vesicle-like particles in the cell wall close to the cuticle. Transition betwixt cell division and elongation was accompanied past a dramatic increase in cuticle thickness, which represented more than one-half of the outer epidermal wall, and the lamellate arrangement of the non-cutinized cell wall. Changes in this non-cutinized outer wall during evolution showed specific features non shared with other cell walls. The coordinated nature of the changes observed in the cuticle and the epidermal jail cell wall indicate a deep interaction between these two supramolecular structures. Hence, the cuticle should exist interpreted within the context of the outer epidermal wall.

The epidermis is the tissue that covers all plant organs. Information technology is composed of thick-walled cells that are strongly adhered to each other and display specific mechanical properties that confer the necessary force to support plant integrity and control plant growth (Kutschera and Niklas, 2007; Savaldi-Goldstein et al., 2007; Javelle et al., 2011). In addition to differences in cell wall thickness between the epidermis and the remainder of inner tissues, in that location is as well asymmetry within epidermal cell walls (Glover, 2000). The outer epidermal wall (OEW), existence the structure that bears most of the stress exerted past growing internal tissues, is considerably thicker than the inner periclinal and anticlinal walls (Glover, 2000; Kutschera, 2008). This disproportion is farther reinforced by the deposition of a lipid-rich and highly hydrophobic cuticle layer.

The establish cuticle is a protective layer that covers the outer wall of the aerial parts of higher plants. Information technology constitutes the chief bulwark between the atmosphere and the plant, thus serving unlike protective functions (Heredia, 2003). In this sense, the cuticle prevents massive water loss, regulates gas exchange, protects against mechanical injury and pathogen invasion, filters potentially damaging UV lite, and generates a microenvironment suitable for certain organisms (Holloway, 1982; Riederer and Müller, 2006; Domínguez et al., 2011; Yeats and Rose, 2013). In the last years, a role for the cuticle in postharvest fruit quality has been manifested (Becker and Knoche, 2012; Lara et al., 2014; Martin and Rose, 2014). Most of these functions have been demonstrated for isolated cuticles; nevertheless, it should non exist forgotten that the epidermis contributes to these properties in planta.

The cuticle has a complex and heterogeneous nature. It is composed of a fraction of soluble waxes (Samuels et al., 2008), mainly devoted to reducing water transport, deposited on and in an insoluble matrix of cutin, which constitutes the primary component of the cuticle. A pocket-size phenolic fraction is also present in the cutin matrix (Hunt and Baker, 1980). Cutin forms an baggy and viscoelastic framework based on the interesterification of C_sixteen and C₁₈ polyhydroxyalkanoic acids (Heredia, 2003; Domínguez et al., 2011). The meaning progress made toward understanding cutin chemical limerick and its monomer biosynthesis (Heredia, 2003; Pollard et al., 2008; Beisson et al., 2012) has recently been accompanied by a partial description of the machinery of transport and further synthesis of cutin (Heredia-Guerrero et al., 2008; Girard et al., 2012; Yeats et al., 2012; Domínguez et al., 2015).

The cuticle membrane lies over and merges into the outer wall of epidermal cells (Martin and Juniper, 1970). Thus, a fraction of cell wall polysaccharides is embedded in the cuticle and tin be regarded as a cuticle component. This polysaccharide fraction present in the isolated cuticle is variable among species and developmental stages, depending on the degree of merging between the cutin matrix and the prison cell wall. Hence, the cuticle tin exist considered a fine modification of the epidermal cell wall or an integral part of it, since it is already present during embryogenesis (Javelle et al., 2011). Again, due to the deep association between the cuticle and the cell wall, they both have overlapping backdrop and functions (Thompson, 2001).

A lot of work has been carried out on the fine structure of the cuticle and cell wall. Jeffree (2006) compiled and analyzed the country-of-the-art data on cuticle fine structure for a large number of plant species. Diverse cuticle morphological and structural types, from lamellate and relatively ordered structures to reticulate and mainly amorphous ones, take been observed in different establish species (Jeffree, 2006). Moreover, changes in cuticle morphology and structure can exist displayed during plant development (Riederer and Schönherr, 1988). This complex ultrastructural scenario needs to be integrated with our still incomplete noesis on the synthesis and assembly of cuticle components, mainly cutin, and their interactions with an evolving jail cell wall. Cell wall ultrastructure has commonly been analyzed with antibodies against unlike polysaccharide components, and mostly performed to study cell wall changes during fruit ripening. Still, the structure and office of growing prison cell walls and their cuticle membranes is still not clear (Fry, 2004; Jeffree, 2006). In order to gather knowledge on the OEW functions, it is important to investigate the modifications and distribution of their components too as their dynamic changes. Despite the significant office of the epidermis and cuticle on plant growth and interaction with the surround, there is deficient literature on their structural and morphological changes during plant evolution.

Tomato fruit has been extensively analyzed at the prison cell wall and cuticle levels due to its economic importance. Pregnant information on the biophysical (Bargel et al., 2006; Domínguez et al., 2011), physiological (Baker et al., 1982; Domínguez et al., 2008), and biochemical (Lunn et al., 2013) properties of the cuticle has been gathered over the years as well equally on the physiological changes occurring during fruit growth and ripening (Seymour et al., 2013). This work studies the morphological and structural modifications of epidermal cells and cuticle during tomato fruit evolution. A report of this nature is also necessary for a better understanding of the formation of the biopolymer cutin. In this sense, in this work we depict a detailed ultrastructural scenario of the genesis and further development of tomato fruit cuticle. Thus, the aim of this research is 2-fold: to describe changes of fine structure of cuticle and epidermal prison cell wall in the form of tomato fruit evolution, and to correlate the already existing information on cuticle chemical composition and biophysical characteristics with its fine structure.

RESULTS

Fig. 1 shows changes in the number of epidermal cells per surface unit of measurement during fruit growth and development. The period of jail cell division corresponded to approximately the first ten days after anthesis (daa) while from 11 daa, cell expansion became prominent. The number of cells per surface expanse did not remain constant during the cell division period only decreased one time fruit was gear up and started its growth (2 daa) and so remained abiding until 8 daa. Two consecutive and pregnant drops in the number of cells were observed at nine and 11 daa. From eleven daa, the number of epidermal cells per surface area slowly decreased until it reached a minimum at ruby ripe. A transition menstruation between cell sectionalisation and cell enlargement was observed at 9 to 10 daa, where a combination of cell partition and jail cell expansion was observed.

Number of epidermal cells per surface unit of measurement (mm²) during Cascada fruit growth. Data are expressed as mean ± se.

Epidermal Fine Structure at Early on Stages of Development

Fig. 2 shows transverse sections of Cascada fruit tissue during the outset six d of organ growth. At anthesis, no tissue differentiation was observed, with all cells showing irregular shapes and sizes. However, the outer wall of protodermal cells, still not differentiated as epidermis, already displayed a higher thickness in comparing to the radial or inner walls (Fig. 2A). These protodermal cells were not arranged in the aforementioned aeroplane, but some cells protruded. At 2 daa, fruit surface began to flatten and parenchyma tissue differentiation was initiated (Supplemental Fig. S1). However, the outermost two to 3 layers of cells still appeared disorganized and undifferentiated, showing dissimilar sizes and morphologies: cubical, triangular, or spherical (Fig. 2, C and D). Cuticle could exist observed from the commencement as a very sparse dark layer roofing the outer walls of protodermal cells. The so-called cuticle ridges, which are ordinarily detected on surface images, could likewise exist observed in the cross sections (run into arrows in Fig. ii, A–C) and corresponded to OEW irregularities, not to local increases of cuticle textile since cuticle thickness was not altered in these areas. At anthesis, these ridges were detected all over the surface (Fig. 2A) while later on they were restricted to the regions close to anticlinal walls (run into arrows in Fig. 2, B and C). They slowly disappeared during the first 5 to six daa, and at 6 daa, the OEW surface was completely flattened (Fig. 2D). Fig. 2, Due east and F, shows scanning electron microscopy images of the tomato fruit surface where information technology could be observed that these ridges did not follow any orientation. The flattening of the surface during this period was also observed by scanning electron microscopy, with cells at anthesis showing a clear curvature and cells at 5 daa flattened. Differentiation of epidermal and hypodermal tissue started at 7 daa and was already finished at 8 daa (Fig. 3A). At this stage, an increase in prison cell size was also observed. Comparing of Fig. 3B (9 daa) and Fig. 3C (ten daa) showed a dramatic increment in cuticle deposition and thickness within a ane d period. This was accompanied by the first of cuticle impregnation of epidermal radial walls initiating the so-chosen pegs (Fig. 3C). The increased cuticle deposition observed at 10 daa connected, and only v d later on (15 daa) the thickness was doubled (Fig. 3D).

Electron microscopy pictures of the epidermis of Cascada fruits during the showtime half-dozen d of growth. TEM (A, B, C, D); Scanning electron microscopy (E, F). Ovary (A), bar 10 μm; two daa (B), bar 2 μm; five daa (C), bar v μm; 6 daa (D), bar x μchiliad; ovary (E), bar 1 μ1000, 10 kV; 5 daa (F), bar 8 μk, 10 kV. Arrows indicate the presence of wrinkles.

TEM pictures of epidermal cross sections of Cascada fruits. 8 daa (A), bar x μm; ix daa (B), bar 5 μm; 10 daa (C), bar ten μthou; fifteen daa (D), bar 5 μm.

Effigy 4 shows the average thickness of both the cuticle, i.due east. cutinized cell wall, and the cell wall of tomato fruit from anthesis until fifteen daa. Cuticle thickness slowly increased during the first 9 d, changing from 60 nm at anthesis to 480 nm at nine daa, while at x daa it reached five.3 μthousand. This sudden escalation in cuticle thickness was very noticeable and marked the start of a period of increasing cuticle thickness that reached 9.4 μm at 15 daa. The cell wall showed a similar pattern with a wearisome increase from anthesis to 9 daa followed by a higher rise in thickness during the ten to xv daa period. It is interesting to note that during the first 9 d menstruum of growth, the thickness of the cell wall was significantly higher than that of the cuticle, while from 10 daa on this tendency was reverted and cuticle thickness remained higher. Jail cell wall thickness was around 7-fold college than cuticle's thickness during the first six d; subsequently increase in cuticle thickness during vii to 9 daa was not accompanied by a similar increase in the cell wall, and this fold-change was reduced to 2.5.

Evolution of cuticle and outer epidermal wall thickness during the early stages of fruit growth. Open circles, cuticle; solid circles, cell wall. Information are expressed every bit mean ± se.

Effigy v shows high-magnification transmission electron microscopy (TEM) images of the cuticle during the cell division flow. The cuticle was observed as a uniformly distributed and electron-dumbo thin layer at anthesis (Fig. 5A). At 2 daa, the inner side of the cuticle started showing a globular morphology (see arrows in Fig. 5B) albeit the cuticle nevertheless appeared as a very homogeneous electron-dense layer. This globular nature of the cuticle's inner side was more than pronounced in later stages (Fig. 5, C–Due east) with the presence of nanoscopic, polydisperse (in size), and electron-dense particles, droplets, or vesicle-like particles randomly distributed in the jail cell-wall region in close contact with the cuticle. Thus, most part of the cuticle displayed a non-homogeneous condition during the 5 to 9 daa menstruum. At 10 daa (Fig. 5F), a reticulate region was observed on the upper half of the cuticle whereas the lower function still displayed a globular nature.

High magnification pictures of the epidermis of Cascada fruits during the prison cell division period. Ovary (A), bar 500 nm; 2 daa (B), bar 200 nm; seven daa (C), bar 500 nm; 7 daa (D), bar 200 nm; 9 daa (E), bar 500 nm; 10 daa (F), bar 500 nm. Arrows signal the location of globular structures.

Epidermal Fine Construction during Cell Expansion

The cuticle and cell wall suffered few changes during cell expansion period. Cuticle thickness continued growing during this menstruation and reached a maximum around 25 daa to afterwards decrease during the last stages of development and ripening (Fig. 6). Prison cell wall thickness also increased during this catamenia until ripening. A plateau during the 15 to forty daa was observed, with no significant changes in thickness, and the increase in thickness was resumed during ripening. Interestingly, around the breaker stage (45 daa), both cuticle and cell wall reached similar thicknesses and remained like until carmine ripe. A transient increase at 50 daa was observed in both the cuticle and the cell wall.

Changes in cuticle and outer epidermal wall thickness throughout fruit growth and ripening. Open circles, cuticle; solid circles, cell wall. Information are expressed as mean ± se.

Figure 7 shows TEM images of transverse epidermal sections. Cuticle pegs were well developed at 20 daa (Fig. 7A); degradation of cuticle cloth on the anticlinal and inner periclinal walls of the epidermis continued throughout this period until red ripe (Fig. 7, C and E). Loftier magnification images of the cuticle during this period showed a homogenous electron-dumbo cuticle layer without stardom betwixt layers with dissimilar osmiophilic beliefs (Fig. 7, B, D, and F). The globular nature of the inner cuticle surface was lost during this period, despite cuticle deposition continuing throughout this catamenia.

TEM pictures of epidermal cantankerous sections of Cascada fruits during cell expansion and ripening. A, B, 20 daa, bar 10 μg (A) and two μ1000 (B); C, D, 35 daa, bar 10 μm (C) and 2 μthou (D); Due east, F, 55 daa, bar ten μm (Eastward) and two μm (F).

Cell wall ultrastructure showed an amorphous, mostly electron-articulate nature at early stages of development (Fig. 8A). At 5 daa, the cell wall started to bear witness a reticulate nature (Fig. 8B). Past ten daa, this reticulate disappeared and an organized multilayered prison cell wall was detected (Fig. 8C). This cell wall structure of parallel layers of electron-dense and electron-articulate textile remained constant until ripening (Fig. 8, D–F).

Jail cell wall ultrastructure of Cascada fruit epidermis. 2 daa (A), bar 0.2 μm; v daa (B), bar 0.five μm; 10 daa (C), bar 1 μthousand; 14 daa (D), bar ane μthousand; 30 daa (E), bar 1 μ1000; 50 daa (F), bar 2 μg.

Immunocytolocalization of Prison cell Wall Components to the OEW

Changes in jail cell wall composition were studied with antibodies against the chief cell wall components: methyl-esterified and non-esterified pectin and cellulose. Table I shows changes in pectin and cellulose labeling density of the cell wall throughout during fruit development. The presence of non-methyl-esterified pectin was restricted to the early stages of development, from ovary to 7 daa (Fig. 9A), and was non detected during the rest of fruit development and ripening. On the contrary, methyl-esterified pectin was detected during the outset stages of development (from ovary until ten daa) (Fig. 9B) and after during ripening with a lower density. During the menstruation of cell expansion, esterified pectins were scarcely detected. Crystalline cellulose was detected in the prison cell wall during fruit growth but was absent-minded during ripening (Fig. 9, C and D). Labeling was not restricted to any specific area of the cell wall merely distributed forth it. No labeling was detected within the cuticle.

Tabular array I.

Pectin and cellulose labeling density during tomato fruit development

Labeling density in the outer epidermal wall is expressed as the number of gold particles per μm².

Phase (daa)	Non-Esterified Pectin	Esterified Pectin	Crystalline Cellulose
Ovary	2.7 ± 0.4	3.9 ± 0.3	3.9 ± 0.4
2	2.7 ± 0.1	3.0 ± 0.4	i.3 ± 0.1
5	2.seven ± 0.seven	4.1 ± 0.3	two.7 ± 0.three
7	two.9 ± 0.4	3.6 ± 0.2	iii.1 ± 0.2
9	—	3.5 ± 0.3	3.2 ± 0.four
x	—	3.four ± 0.2	3.vii ± 0.3
xv	—	—	3.1 ± 0.iii
xx	—	—	iv.five ± 0.3
25	—	—	5.5 ± 0.v
35	—	—	v.5 ± 0.8
45	—	two.4 ± 0.1	—
55	—	2.2 ± 0.three	—

Data are presented equally mean ± se. Controls without primary antibodies showed label density < 0.3. daa, days later anthesis; (—), no labeling or very disperse gold particles.

Immunolocalization of cell wall components in epidermal cantankerous sections of Cascada fruits. 7 daa non-esterified pectin (A); 5 daa esterified pectin (B); ovary crystalline cellulose (C); 35 daa crystalline cellulose (D). Gilt particles are encircled in crimson. Bars = 200 nm.

DISCUSSION

According to the standard and prevalent nomenclature, the plant cuticle consists of ii different layers: the cuticle proper equally the outermost region that appears at early stages of development, and the underlying cuticular layer that appears after in development past cutin impregnation of a portion of jail cell wall (Jeffree, 2006). Thus, the more than electron-dumbo cuticle proper is mainly composed of cutin and is complimentary of polysaccharides, whereas the cuticular layer also contains polysaccharides derived from the cell wall (Jeffree, 2006). In this work we have omitted this classification for several reasons. Tomato fruit cuticle does not show regions of unlike electron-density but appears as a more or less homogeneous osmiophilic layer. Moreover, contrary to other species such every bit Agave americana 50. and Picea abies (L.) H. Karst (Jeffree, 2006), no clear boundary between a fibril-rich and non-fibril region can be detected inside the cuticle. In this sense, OEW localization of jail cell wall components with antibodies failed to place their presence in the cuticle, most probably because its ascendant lipid nature hindered epitope recognition. Epitope masking has been reported to hinder recognition of prison cell wall components (Marcus et al., 2008). Therefore, nosotros simply differentiate two regions in the OEW: the cuticle (or cutinized jail cell wall) and the cell wall (or non-cutinized jail cell wall), based on the presence or absenteeism of osmiophilic material of lipid nature.

Tissue Differentiation

In the short menstruum spanning between anthesis and 2 daa, fertilization had occurred and fruit set had been initiated. This was conspicuously observed from the pregnant change in fruit size (almost doubling) that happened during this period, plus the histological changes observed in cross sections. At 2 daa, parenchyma differentiation had started and cells had increased their size and initiated a layered arrangement. However, the beginning three layers, which correspond to the future epidermis and collenchyma, remained undifferentiated. Epidermis and collenchyma differentiation did not start until 7 daa, before the beginning of the prison cell expansion period. Despite organization of the epidermal layer and prison cell arrangement, which did non start until 7 daa, the most distinguishing feature of epidermal cells, a thick and cutinized OEW, was already present in the ovary.

Organs exhibit specific spatial and temporal patterns of growth (Anastasiou and Lenhard, 2007). This growth is a combination of two integrated processes, cell division and cell expansion. Whereas the offset is mostly limited to early on stages of evolution, cell expansion is responsible for most of an organ's growth and mainly occurs after jail cell division has ceased. In blood-red tomatoes, transition between sectionalization and expansion occurs around 11 to fourteen daa (Bertin et al., 2007; España et al., 2014). Several transcriptional factors involved in the regulation of cell division and expansion have been identified (Anastasiou and Lenhard, 2007). Recently, changes in cuticle mechanical backdrop were associated with the transition between jail cell division and expansion (España et al., 2014). Results reported hither also showed a temporal correlation among changes in OEW morphological and biochemical traits and the transition betwixt these two processes of organ growth. During cell partitioning, the OEW was thin and generally composed of non-cutinized cell wall rich in non-esterified and esterified pectin whereas the transition to cell enlargement was accompanied by a dramatic increase of the OEW'due south thickness due to cuticle aggregating, and pectin epitopes were barely detected in the remaining not-cutinized cell wall. Thus, during prison cell expansion the cuticle represented sixty% to 75% of the OEW thickness. Fujino and Itoh (1998) also observed a significant increase in OEW thickness in pea stems between elongating and non-elongating cells. In this sense, cuticle thickness was 4-fold college in elongating cells whereas cell wall only showed a 2.v-fold. Recently, it was reported how changes in the cutin/polysaccharide ratio present in the cuticle afflicted the strength required to achieve viscoelastic deformation, and thus growth (España et al., 2014). At the whole OEW level, it would be most interesting to investigate whether the dramatic change in thickness of the cuticle/prison cell-wall ratio observed during the transition between cell division and elongation plays a similar mechanical role.

The Discontinuous Construction of the Cutinized Cell Wall

At anthesis, fruit surface showed the presence of multiple wrinkles without whatever clear orientation that disappeared during the next v d of fruit growth. These cuticle folds take besides been observed in Vitis vinifera L. fruits (Considine and Knox, 1979; Casado and Heredia, 2001) and in the leaves of apple tree (Bringe, 2007), Aesculus hippocastanum L. (Martin and Juniper, 1970), Syringa vulgaris L. (Holloway, 1971), and Acer pseudoplatanus 50. (Wilson, 1984) amongst other species. However, their evolution has just been characterized in grape and apple fruits, with surface flattening observed during organ development (Casado and Heredia, 2001; Bringe, 2007). Co-ordinate to Rosenquist and Morrison (1988), these folds represent storage of cuticle textile that spread out during the period of rapid growth. However, epidermal cross sections showed that these folds are not only composed of cuticle, but they represent an irregular jail cell-wall profile covered by a cuticle. The fact that these wrinkles began to disappear the moment fruit growth started, that epidermal surface was completely apartment at half dozen daa, when only ten% of fruit growth has occurred, and that they were already present prior to anthesis (Supplemental Fig. S2) suggests that these folds could play a unlike office, perchance related to the irregular protodermal surface nowadays in the ovary. As Jeffree (1996) pointed out, the presence of folds increases surface area by a gene of 2 to 3. These wrinkles probably play a different part to those found on the surfaces of full developed petals (Jeffree, 2006) and referred to as ridges or nanoridges (Li-Beisson et al., 2009).

Changes associated with the beginning of fruit ready were also detected at the cuticle level. At anthesis, the cuticle appeared as a very narrow but well-divers electron-dense layer. Ii days later, the cuticle doubled its thickness, although information technology remained as a very sparse layer with a globular and irregular inner surface visible. Until 9 daa, the cuticle moderately increased its thickness and the globular contour of its inner face up in contact with the jail cell wall was increasingly manifest. Following the literature on cuticle ultrastructure, this thin layer present until 9 daa can be regarded as the procuticle (Jeffree, 2006). The procuticle is considered an early stage of cuticle development and it is believed to be composed of cutin only. According to Jeffree (2006), the procuticle is underlain past a superficial layer that behaves as a polyanion and is predominantly pectic in nature (Jeffree, 2006). The procuticle has been identified in only a few species (see references in Jeffree, 2006) and in this piece of work, to our knowledge, is reported for the first time in tomato fruit. The variable shape, by and large of its inner side, and thickness of this procuticle (Fig. 5, C–E) is probably a consequence of the progressive molecular organisation and assembly of cutin molecular domains that happens at an early growth stage. The globules in contact with the inner side of the procuticle were also observed as small osmiophilic droplets and globular-like particles along the underlying cell wall (Fig. v, D and E). These globular-like particles, of varying size, seem to migrate thorough the wall matrix to the cuticle. This fact has been conspicuously observed by several authors and these particles accept been considered procutin precursors (Frey-Wyssling and Mühlenthaler, 1965; Hallam, 1970; Heide-Jørgensen, 1978, 1991; Jeffree, 2006). In this study there were no clear indications of where this lipid fabric is synthetized or how information technology is transported to the surface of protodermal cells. Nevertheless, accumulation and fusion of the above-mentioned nanodroplets or nanoparticles at the outer part of epidermal plant cell wall has been suggested equally a possible mechanism for early cutin or procuticle germination (Heredia-Guerrero et al., 2008; Domínguez et al., 2010; D'Angeli et al., 2013; Kwiatkowska et al., 2014). During the prison cell expansion period (from effectually eleven daa until red ripe), the cuticle showed few changes in appearance and fine structure. It could exist said then that the main framework has already been constructed and the only observable changes during this period were further development of invaginations between neighboring epidermal cells and cuticle thickening most probably due to rearrangement of the lipid matrix, since the amount of isolated cutin did not alter during this period (Domínguez et al., 2008).

Cuticle development showed 2 marked changes in thickness. During the first ix d, cuticle thickness was within the nanometric calibration, whereas at 10 daa, due to a sudden 11-fold increment, it reached the micrometric scale. This modify in cuticle thickness agrees with the results previously reported for the same cultivar using selective dyes and conventional microscopy and was accompanied by a 4-fold increase in cuticle material (Domínguez et al., 2008). It is interesting to note that, although the thickness values herein obtained were similar to those reported by Domínguez et al. (2008), at that place is a significant discrepancy at ten daa. Values reported here are higher probably because osmium is more sensitive to depression quantities of lipid material than Sudan 4. Cuticle thickness continued rising until 25 daa, followed by a decrease during the ripening period. This decrease in thickness was not accompanied by a loss of cuticle textile, which suggests a rearrangement of cuticle material during this period.

The Growing Non-Cutinized Jail cell Wall: a Circuitous and Heterogeneous Construction

The polysaccharide prison cell wall, which serves as a framework for cuticle deposition and development, is as well a dynamic supramolecular structure in continuous change during growth. A significant increase in thickness was observed throughout development, although much lower than in the case of the cuticle. This alter in thickness was more prominent during the jail cell division stage when the cell wall showed a 4-fold increase in a very brusk period of time. Later on, this growth continued at a slower footstep until 20 daa. From this menstruum until the end of ripening, the cell wall did not change its thickness except for a transient increase during ripening. Cell wall swelling is known to occur during fruit ripening in several species (Redgwell et al., 1997).

Most studies on cuticle development are non accompanied past an examination of the accompanying cell wall, with some exceptions (run across Jeffree, 2006). On the other hand, only a few studies on cell wall changes during development are focused on the OEW, since it is considered a special prison cell wall, not comparable to other cell walls. Despite the significant number of studies on jail cell wall limerick and analysis, isolation and further assay of the OEW is not trivial. Microscopic analysis of the OEW of pea stems showed college cellulose content than typical main walls (Bret-Harte and Talbott, 1993). Isolation of the polysaccharide fraction of mature tomato fruit cuticles showed an equimolecular contribution of the three master components of jail cell wall: cellulose, hemicellulose, and pectin (López-Casado et al., 2007). Recently, using enzyme-gilded labeling, Guzmán et al. (2014) reported the identification and location of cellulose and pectin in isolated cuticles of Eucalyptus L'Hér., Populus L., and Pyrus 50. leaves. In this work, results obtained by the use of antibodies confronting the ordered non-esterified pectin, the gelatinous esterified pectin, and crystalline cellulose allowed us to draw the following molecular scenario. Crystalline cellulose was nowadays in the cell wall throughout development and just disappeared during ripening. This is in agreement with the biochemical analyses of tomato jail cell walls where the corporeality of cellulose remained constant during evolution and only decreased during ripening (Lunn et al., 2013). The presence of non-esterified pectin was constrained to early stages of development, the cell division menses, and was not located during the expansion flow. Esterified pectin, which forms role of the bulk of polysaccharides that etch the gel-like matrix of the walls (Burton et al., 2010), was constitute with a moderate and notable density during prison cell partition, and later on during ripening. The scarce presence of methyl-esterified pectin during most of the cell expansion period could be due to a reduction of the molecules, merely the possibility of epitope masking during this period should also be considered (Marcus et al., 2008).

Several studies point to a role of pectin methyl-esterification in prison cell wall expansion. Thus, immunocytochemical analyses of root cells showed that dividing cells contained lower levels of not-esterified pectins whereas no differences in esterified pectins were observed between dividing and non-dividing cells (Dolan et al., 1997). Similarly, elongating epidermal cells have less non-esterified pectin than non-elongating cells (Fujino and Itoh, 1998). Low pectin methyl-esterification has besides been shown to decrease prison cell elongation in several cases (Derbyshire et al., 2007; Guénin et al., 2011), although the opposite behavior has also been reported (Pelletier et al., 2010). It has been suggested that local distribution of jail cell wall polysaccharides, mainly pectins, could also play an important office in prison cell wall physico-chemical properties (Levesque-Tremblay et al., 2015). In this sense, a heterogeneous transversal distribution of esterified and non-esterified pectins in the OEW of flax and pea was found, with non-esterified pectins mainly located to the outer part of the OEW and esterified pectins more arable in the inner part (Jauneau et al., 1997; Fujino and Itoh, 1998). Our results, however, did not show any distribution pattern for any of the cell wall polymers studied.

Information technology is interesting to note the change in cell wall ultrastructure that appeared during the onset of cell expansion, with parallel multilayers disposed in a helicoidal class (Fig. viii). This organization agrees well with the structure of the OEW discussed by Kutschera (2008) and is equanimous of an ordered pattern with helicoidally organized layers of inextensible cellulose microfibrils. This pattern has been suggested to exist the event of a well-organized self-assembly mechanism (Rey, 2010).

Many studies have been conducted to analyze the cell wall modifications that accompany fruit ripening (Brummell, 2006). In lycopersicon esculentum, a decrease in cellulose and an increase in non-esterified pectin are observed during ripening (Roy et al., 1992; Steele et al., 1997; Lunn et al., 2013). However, these studies have been carried out in parenchyma cell walls simply not in the OEW. Our results suggest a slightly different behavior in the OEW: a decrease in crystalline cellulose during ripening, and an increase in esterified pectins.

CONCLUSIONS

Results herein presented showed that the OEW is a highly dynamic structure that is significantly modified during the early on stages of fruit growth. A deep interconnection and coordination between the two supramolecular structures that compose the OEW, i.e. the polyester cutin and cell wall polysaccharides, was observed. Farther research will be necessary to investigate the molecular signals that trigger this synchronized development. In view of these results, the institute cuticle should be explained within the epidermis framework. In this sense, cuticle mutants would be an splendid material to report the interaction between the epidermis and the cuticle and could shed new light on the role of several cuticle genes. Given the significant role of the epidermis in organ development and fruit quality, these results will exist valuable to create new commercial varieties with desired traits. Finally, this work (to our knowledge) shows for the first time the importance of the early on stages of fruit evolution in cutin arrangement and deposition—a catamenia of time that any proposed mechanism of cutin synthesis should have into careful consideration.

MATERIALS AND METHODS

Plant Textile

Solanum lycopersicum L. Cascada plants were grown in a polyethylene greenhouse during spring. A random design of iii blocks with 10 plants per cake was employed. Experiments were conducted at the Estación Experimental La Mayora, Consejo Superior de Investigaciones Científicas, in Málaga, Spain. Tomato seedlings were grown in an insect-proof glass house, and plants were and so transplanted to soil in a plastic house at the three-truthful-foliage-growth stage. The within-row and between-row spacing was 0.5 and 1.5 g, respectively. Plants were watered when necessary using a nutrient solution (Cánovas, 1995) and were supported by cord and pruned to a single stalk. The harvesting period lasted from early May until center July. Flowers were labeled at anthesis and fruits collected at selected time points, from anthesis to ruby-red ripe (see Fig. 10). Fruit diameter was measured with a caliper from a minimum of 100 fruits (Supplemental Fig. S3).

Photograph of Cascada fruits at the different stages of development studied in this work. Numbers indicate days after anthesis; 0 refers to anthesis. Bar = i cm.

Tissue Sectioning and Immunolabeling for TEM

Three fruits per developmental stage were harvested and minor pericarp pieces of each fruit were cut and stock-still overnight with four% paraformaldehyde in 0.1 m phosphate buffer (pH seven.four), rinsed in the same buffer and postfixed in 1% OsO_four in distilled water for one h. Afterward dehydration in a graded ethanol-water series from fifty to 100%, the samples were embedded in LR White resin and polymerized in BEEM capsules for 48 h at 65°C. Ultrathin sections (around fifty nm thick) were cut with a Reichert Jung Ultracut ultra-microtome (Reichert Technologies, Vienna, Austria) using a glass knife. Sections were placed onto nickel-Formvar-coated grids and examined in a JEOL JEM-1400 (Akishima, Tokyo, Japan) TEM at 80-kV acceleration voltage.

Prior to visualization, some samples were labeled with different antibodies to report cell wall structure. Sections were treated with 10% hydrogen peroxide for 15 min to remove osmium and washed in distilled h2o and in phosphate-buffered saline (PBS) buffer (0.01 M, pH vii.4) (Kwiatkowska et al., 2013). Samples were then blocked with modest drops of PBS buffer containing three% bovine serum albumin for i h at room temperature and after washed iii times in PBS buffer. Rat monoclonal antibodies LM19 and LM20 (www.plantprobes.net) were employed to characterization not-esterified and methyl-esterified homogalacturonan domains of pectin, respectively. Sections were incubated in a 10-fold dilution of LM19 or LM20 in PBS buffer for 1.5 h. Afterward a series of three washes in PBS buffer, grids were incubated in a fifty-fold dilution of goat anti-mouse IgG coupled to 15 nm diameter colloidal gold particles (Aurion, Wageningen, The netherlands) and incubated at room temperature for i h. His-tagged recombinant CBM3a (saccharide-binding module) protein (world wide web.plantprobes.net) was employed to detect crystalline cellulose (Blake et al., 2006). Sections were incubated in a 100-fold dilution of CBM3a in PBS buffer with 0.1% bovine serum albumin for 1 h at room temperature. Samples were then incubated in a 100-fold dilution of mouse anti-His monoclonal antibody (Sigma-Aldrich, St. Louis, MO) in PBS buffer for 1.v h. After three washes in PBS buffer, grids were incubated in the same secondary antibiotic every bit sections immunolabeled with LM19 and LM20. Afterward a thorough wash in distilled water, all sections were stained with a 4% uranyl acetate solution for 15 min and later on on carefully washed with distilled h2o. Controls without primary or secondary antibodies were also carried out.

Scanning Electron Microscopy and Epidermal Cell Number

Pocket-sized pieces of pericarp from three biological samples at each phase of development were stock-still in 2.5% glutaraldehyde in PBS buffer pH 7.ii, and afterward on dehydrated in an ethanol serial and their surfaces inspected with a scanning electron microscope (model no. JSM-840; JEOL). V images from each biological sample were taken and the number of cells per surface area counted using ImageJ (Rasband, 1997–2014).

Statistics

Cuticle and jail cell wall thickness measurements were estimated from the cantankerous-sectioned samples at each phase using an prototype capture analysis program (Visilog v. vi.3; Noesis, Crolles, France). It was assessed from the primal region between pegs every bit this expanse remains almost constant and is not affected by cuticle invaginations. The density of labeling, i.east. the number of gold grains per unit area (μm^two), was estimated using the aforementioned program. Thirty micrographs were analyzed for each stage of development. Data are expressed as mean ± se.

Supplementary Textile

Acknowledgments

Nosotros give thanks the Electron Microscopy Facility at the Universidad de Málaga for helpful assistance.

Notes

Glossary

OEW	outer epidermal wall
daa	days later anthesis
dba	days before anthesis

Footnotes

¹This piece of work was partially supported by grant no. AGL2012-32613 of the Plan Nacional de I+D, Ministry of Pedagogy and Science, Spain.

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