Development of fruit cuticle in cherry tomato ... - CSIRO Publishing

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Functional Plant Biology, 2008, 35, 403--411

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Development of fruit cuticle in cherry tomato (Solanum lycopersicum) Eva DomínguezA, Gloria López-CasadoB,C, Jesús CuarteroA and Antonio HerediaB,D Estación Experimental ‘La Mayora’ (CSIC) Algarrobo-Costa, E-29750 Málaga, Spain. Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, E-29071 Málaga, Spain. C Present address: Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA. D Corresponding author. Email: [email protected] A B

Abstract. The cuticle of a plant plays an important role in many physiological events of fruit development and ripening. Despite this, little is known about cuticle formation and development. We include a detailed morphological study at the microscopic level of cuticle during fruit growth and ripening using tomato as a fruit model. In addition, a study of the differences in cuticle thickness and composition during development is included. The four genotypes studied in this work showed a similar timing of the main morphological events: initiation of epidermal differentiation, changes in the distribution of the lipid, pectin and cellulose material within the cuticle, appearance of pegs, beginning of cuticle invaginations, maximum thickness and loss of polysaccharidic material. Fruit growth, measured by fruit diameter, showed a positive correlation with the increase of cuticle thickness and the amount of cuticle and their cutin and polysaccharide components per fruit unit during development. By contrast, cuticle waxes showed a different behaviour. Two important characteristics of cuticle growth were observed during tomato fruit development. First, the amount of cuticle per surface area reached its maximum in the first 15 days after anthesis and remained more or less constant until ripening. Second, there was a significant loss of polysaccharidic material from the beginning of ripening (breaker stage) to full red ripe. Additional keywords: cuticle components, cuticle growth, cutin, epidermis morphology, tomato fruit.

Introduction Aerial parts of higher plants are covered by a continuous extracellular layer -- the cuticle. The main functions ascribed to the cuticle are to minimise water loss, to limit the loss of substances from plant internal tissues and also to protect the plant against physical, chemical and biological impacts (Jeffree 2006). Cuticles of higher plants are chemically heterogeneous in nature, consisting of a wax fraction, soluble in common organic solvents, and an insoluble cuticular matrix, that forms the framework of the cuticle. This cuticular matrix is mainly formed by the biopolymer cutin, a high-molecular weight polyester composed of various inter-esterified C16 and C18 hydroxyalkanoic acids (Walton 1990; Heredia 2003). Removing waxes and cutin from isolated cuticles yields some residual material, predominantly polysaccharides, that represents the portion of the epidermal cell wall to which the cuticular membrane was attached (Jeffree 2006). Recently, Jeffree (2006) proposed a detailed model for the construction, arrangement and assembly of the different components of a standard cuticle during ontogeny, assuming that the cuticle of leaves and fruits undergo very large changes in area as cells divide and expand during organ growth until late in development. These changes must be reflected in the morphology and accumulation of cuticle components. However, only a few studies have been focussed on the morphological changes of the epidermal cells and the microscopical structure of the cuticle, together with the analysis of the main cuticle components during CSIRO 2008

growth. This limited amount of research on the subject hinders the drawing of general conclusions that could be applied to any plant cuticle. Thus, Riederer and Schönherr (1988) reported the fine structure and cutin composition of Clivia miniata L. leaves during development. Baker et al. (1982) studied the chemical composition and the amount of the different cuticle components during tomato (Solanum lycopersicum L.) fruit growth, and Casado and Heredia (2001) reported a study on the ultrastructure of grape (Vitis vinifera L.) cuticle during fruit growth. More recently, Peschel et al. (2007) have investigated the composition of the cuticle in developing sweet cherry (Prunus avium L.) fruits. These works reported a slow deposition of waxes and cutin during growth together with significant morphological changes at the microscopic level (Riederer and Schönherr 1988; Casado and Heredia 2001). Understanding the detailed process of cuticle formation is important to determine the stages in which significant cuticular changes happen as well as the time-frames in which genes related to cuticle synthesis might be especially active. Furthermore, it would be useful to determine the physiological stages of development on which changes in environmental factors such as temperature, light and relative humidity could affect the cuticle. This is especially important for tomato, a crop of high economical interest and the model to study fleshy fruit development. In the past few years, cherry tomato has become a product demanded by consumers due to its firm texture and organoleptic qualities. Hence, research has been focussed on the study of this

10.1071/FP08018

1445-4408/08/050403

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Functional Plant Biology

E. Domínguez et al.

type of tomato and some associated physiological disorders such as cracking and sunscald (Bargel et al. 2006). Fruit cuticle can be considered one of the most important targets to study physiopathies such as fruit cracking, which commonly affect cherry tomato (Matas et al. 2004a, 2005). Consequently, research is required on cuticle changes occurring at the morphological and compositional level during fruit development. The objectives of the present study were, therefore, to describe in detail the morphological changes at the microscopical level of the cuticle of cherry tomato fruit during growth, and to characterise the developmental changes in cuticle components (waxes, cutin and polysaccharides) of the corresponding isolated cuticles. Materials and methods Plant material Solanum lycopersicum L. plants cv. Gardener’s Delight, Cornell Inbred 10, Cascada and a recombinant inbred line (RIL) RIL115 were used in this study. The RIL line is an F8 from a population of 164 RILs developed by single seed descent from the interspecific cross ‘Moneymaker’ (S. lycopersicum) ‘TO-937’ (Solanum pimpinellifolium L.). Seeds of Cornell Inbred 10 were kindly provided by Edward Cobb (Cornell University, Ithaca, USA). RIL115 and Gardener’s Delight were selected because of its high incidence of fruit cracking in greenhouse cultivation and Cornell Inbred 10 and Cascada were selected for the opposite behaviour, low fruit cracking. Ten plants of RIL115, Cornell Inbred 10 and Gardener’s Delight together with 40 plants of Cascada were grown in a polyethylene greenhouse during spring 2006. Experiments were conducted at the Estación Experimental La Mayora, CSIC, in the south-east of Spain. Tomato seedlings were grown in an insect-proof glasshouse, and plants of each tomato genotype were then transplanted to soil in a plastic-house at the four-true leaf growth stage. The within-row and between-row spacing was 0.5 and 1.5 m, respectively. Plants were watered when necessary using a nutrient solution (Cánovas 1995) and were supported by string and pruned to a single stem. The harvesting period lasted from early May until mid July. The maximum, minimum and mean temperature, relative humidity and the photosynthetic photon flux density during the growing period are shown in Table 1.

Flowers were labelled at anthesis and fruits collected every 5 days from anthesis to red ripe. To estimate fruit diameter during growth a minimum of 25 fruit per genotype and developmental stage were measured. Fruits with sizes far from the average were not collected. To ensure the isolation of 0.5--1 g of cuticle from Cascada, especially in the first stages of development, 300 fruits were collected at 10 days after anthesis (daa), 150 fruits at 15 daa, 100 fruits at 20 daa, and 40--50 fruits per stage were collected from 25 daa up to red ripe. In spring 2007, 20 plants of Cascada, Gardener’s Delight, Cornell Inbred 10 and RIL115 were grown in the same polyethylene greenhouse following the same cultural conditions. Flowers were also labelled at anthesis and collected at 15 daa, breaker and red ripe stage. One hundred fruits at 15 daa and 50 fruits at breaker and red ripe were collected from each genotype. The temperature, relative humidity and photosynthetic photon flux density during the growing season are shown in Table 1. Tissue sectioning and staining Three fruits of each genotype and developmental stage were harvested and small pericarp pieces of each fruit fixed in a formaldehyde, acetic acid and ethanol solution (1 : 1 : 18); later on dehydrated in an ethanol dilution series (70--95%) and embedded in a commercial resin (Leica Historesin Embedding Kit, Heidelberg, Germany). Samples were cross-sectioned into slices 4 mm thick using a Leica (Wetzlar, Germany) microtome (RM2125). Sudan IV was employed to differentially stain the cuticle using the protocol described by Jensen (1962), Ruthenium Red was used to dye pectin material according to Considine and Knox (1979) and Calcofluor to stain cellulose (Luza et al. 1992). A minimum of five slices per sample were then inspected under light (Sudan IV, Ruthenium Red) and fluorescence (Calcofluor White) microscope (Nikon, Eclipse E800, Tokyo, Japan). Microphotographs were taken with a Nikon camera (DXM1200) coupled to the microscope. Cuticle thickness was estimated from a minimum of 30--50 measurements using an image capture analysis program (VisilogNoesis 6.3). Central region between pegs of the cuticle covering epidermal cells was used to estimate cuticle thickness since this

Table 1. Temperature (8C), relative humidity (%) and photosynthetic photon flux density (PPFD mol m 2 day 1) in the greenhouse during the growing and harvesting period in 2006 and 2007 Data are presented as average minimum, mean and maximum for temperature and relative humidity and mean PPFD Month

Temperature (C) mean maximum

Relative humidity (%) minimum mean maximum

PPFD  (mol m 2 day 1) mean

minimum

April May June July

27 4 25 8 30 4 21 4

14 1 16 2 18 2 20 2

2006 20 2 22 2 25 2 27 1

29 5 30 3 33 2 35 1

36 8 40 12 37 6 40 7

64 8 67 11 64 5 64 5

85 8 90 5 89 3 86 4

April May June

21 8 30 7 30 4

12 1 13 2 17 1

2007 19 2 21 3 23 1

25 3 29 4 30 1

54 11 51 13 57 5

74 6 73 8 76 3

95 3 95 5 95 2

Tomato cuticle growth


area remains almost constant throughout a fruit or leaf and is not affected by cuticle invaginations. Cuticular area was calculated using the same program and a minimum of 20 measures per genotype and stage.

Cuticle components Epicuticular waxes were removed and gravimetically determined after dipping isolated cuticles in chloroform for 10 s at room temperature. In other set of experiments, total cuticular waxes were removed by heating at 50C the isolated cuticles in chloroform : methanol (2 : 1 v/v) for 2 h. Intracuticular waxes were estimated as the difference between total and epicuticular waxes. Cutin isolates were obtained by refluxing dewaxed cuticles in a 6 M HCl solution for 12 h. This procedure removed polar hydrolysable components, mainly polysaccharides (Matas et al. 2004b). Cuticular components (waxes, cutin and polysaccharides) were estimated from two samples per genotype. Statistics Data on fruit diameter, cuticle thickness, cuticular area, and microgram per unit of cuticular area are expressed as means s.e. The number of replicates for each determination is indicated in Materials and methods and also in the corresponding figures and/or tables.

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Cuticle isolation Cuticles were enzymatically isolated from tomato fruits at different stages of development following the protocol by Orgell (1955) as modified by Yamada et al. (1964; see Petracek and Bukovac 1995) using an aqueous solution of a mixture of fungal cellulase (0.2% w/v, Sigma-Aldrich, St Louis, MO, USA) and pectinase (2.0% w/v, Sigma), and 1 mM NaN3 to prevent microbial growth, in sodium citrate buffer (50 mM, pH 3.7). Vacuum was used to facilitate enzyme penetration, and fruit samples were incubated with continuous agitation at 35C for at least 14 days. The cuticle was then separated from the epidermis, rinsed in distilled water and stored under dry conditions.

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Days after anthesis Fig. 1. (A) Evolution of fruit growth, measured as equatorial diameter (mm), from anthesis until red ripe of four tomato genotypes. Data presented as means s.e. of a minimum of 25 fruit per developmental stage and genotype. (B) Evolution of fruit cuticle thickness from 10 days after anthesis until red ripe in the same four tomato genotypes. Data are presented in microns as means s.e. of a minimum of 30--50 measures per genotype and stage.

Results Fruit growth and development Tomato fruit growth, expressed as enlargement in equatorial diameter, showed two phases, one of continuous increase in size and a plateau (Fig. 1A). This plateau is reached on or just before the breaker stage of development. The curve of fruit growth did not show the characteristic sigmoid shape, typical of most tomatoes (Ho and Hewitt 1986), owing to the absence of an initial lag phase. Gardener’s Delight, Cornell Inbred 10 and RIL115 reached the breaker stage at the same time, around 35 daa. The fruits of Cascada ripened later, the breaker stage being attained around 45 daa. Morphological characterisation of the cuticle and epidermal tissue during fruit growth and development The four genotypes studied showed a similar morphological development of the fruit and the associated cuticle. Figure 2

shows in detail some remarkable aspects of the epidermis and the cuticle development. At anthesis, cells were still undifferentiated and showed cubical or spherical morphology with thin cell walls. The cuticle was also very thin and positively stained for lipids (Fig. 2A), pectin staining was too faint and, therefore, not possible to confidently say it was present in the cuticle. Calcofluor gave very weak staining of the cell walls at the first stages of development (Fig. 2G). At 5 daa there was an increase in cuticle thickness (Fig. 2B) and positive staining was observed for lipids and pectins (Fig. 2B, M). Epidermal cells were already differentiated and cells displayed more solid walls. This cell wall thickening continued as the fruit grew. At 10 daa, together with cell enlargement and clear differentiation of parenchymal tissue, three hypodermal layers underneath the epidermis were developed. The cuticle showed wrinkles that could be due to the synthesis of extra lipid material (Fig. 2C, O, P). Sudan IV stained the whole cuticle (Fig. 2C), and

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Fig. 2. Light microscopy photographs of fruit epidermis from four genotypes of tomato at several stages of development. (A--F) Cascada stained with Sudan IV to visualise the cuticle. (A) Anthesis 60, (B) 5 days after anthesis (daa) 60, (C) 10 daa 60, (D) 15 daa 40, (E) 20 daa 40, (F) 35 daa 40. (G, H) Cascada stained with Calcofluor to visualise polysaccharides. (G) Anthesis 60, (H) 15 daa 40. (I--L) Comparative photographs of Cascada. (I), Gardener’s Delight (J), Cornell Inbred 10 (K) and RIL115 (L) at red ripe stage stained with Sudan IV to visualise the cuticle (40). (M, N) Ruthenium Red staining of pectin material in (M) Gardener’s Delight at 5 daa 60, and (N) RIL115 at 15 daa 40. (O, P) Sudan IV staining of 10 daa epidermal tissue from (O) Cornell Inbred 10 40, and (P) RIL115 60. Scale bars: (A--C, G, M, P) 20 mm and (D--F, H--L, N, O) 40 mm.


Composition of fruit cuticle throughout development Of the four genotypes examined in this work, Cascada was chosen to study in full detail cuticle development because plants were vigorous, produced many flowers and had a fruit set of nearly 100%. Any attempts to isolate cuticles from fruits of 5 daa or younger were unsuccessful because the cuticles were too thin and fragile. Figure 3 shows the weight per surface area of isolated Cascada fruit cuticles at different stages of development and of its three main components: cutin, polysaccharides (Fig. 3A) and waxes

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cellulose only appeared discontinuously in the inner cuticle face (Fig. 2H). At 15 daa, the pectin material could be clearly observed in the inner side of the cuticle, together with the cellulosic material (Fig. 2H, N). Lipids were homogeneously distributed throughout the whole cuticle, and heavily impregnated the radial walls of the epidermal cells (Fig. 2D) forming the so-called pegs (Jeffree 2006). At 20 daa, the cuticle continued surrounding the epidermal cell walls, forming cuticle invaginations, and this process continued until ~35 daa (Fig. 2E, F), the number of subepidermal cell layers surrounded by cuticular material and the degree of this invagination being genotype dependent. In the case of Cascada, invaginations partially encircled the first layer of epidermal cell walls, similarly to RIL115 and Gardener’s Delight (Fig. 2I, J, L), although the latter sometimes displayed fragments of cuticular material in the first hypodermal layer. In Cornell Inbred 10, the cuticle extended to the first layer of hypodermal cells which was very incompletely covered (Fig. 2K). The isolated cuticle appeared mainly coloured when the fruit reached the breaker stage, 45 daa for Cascada and 35 daa for the rest of the genotypes. The evolution of cuticle thickness throughout fruit development, from 10 daa to red ripe, was studied in detail for the four genotypes herein investigated (Fig. 1B). As shown by the Cascada pictures, the cuticle of ovaries and fruits of 5 daa were too thin for it to be confidently measured. Similar accumulation timing was observed between fruit growth and cuticle thickness. Cuticle thickness dramatically increased in the first 15 days and then continued growing in a more genotype dependent fashion (Fig. 1B). In Gardener’s Delight there was a decrease in cuticle thickness of 2 mm between 30 and 35 daa (breaker stage) which was accompanied by the last increase in fruit growth at 35 daa (see Fig. 1A). Cuticle thickness then remained stable until red ripe (45 daa). In RIL115 the reduction in cuticle thickness was continuous and more pronounced, 4 mm between 25 daa and red ripe (45 daa). In Cornell Inbred 10 the last step of fruit growth was also observed between 30 and 35 daa but the cuticle thickness remained constant until 40 daa and then decreased, reaching a minimum at the red ripe stage (45 daa). In Cascada maximum cuticle thickness and maximum fruit growth coincided at 40 daa, later on the cuticle diminished 1 mm in thickness in the period between 40 daa and red ripe (55 daa). Thus, there was a general trend towards a decrease in cuticle thickness right after the fruit reached its maximum expansion (around the breaker stage) or at the same time (mature green stage).


Total waxes Epicuticular Intracuticular

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Days after anthesis Fig. 3. Micrograms per square centimeter throughout tomato fruit development of (A) Cascada isolated cuticle and its components cutin and polysaccharides, and (B) Cascada epicuticular, intracuticular and total waxes. Data are presented as means s.e. of 20 measurements per developmental stage.

(Fig. 3B). We note that the percentages of the three main cuticle components remained almost identical during fruit development (data not shown). No correlation between fruit growth and the accumulation of cuticle or any of their components, cutin, polysaccharides or waxes, per fruit area could be observed. Between 10 and 15 daa there was a 3-fold increase in the amount of waxes (Fig. 3B) and a 4-fold increase in polysaccharidic material, cutin and cuticle (Fig. 3A). At 15 daa the net increase in polysaccharides, cutin and cuticle slowed down and almost ceased, although a slight increase was observed until 25 daa. From this stage on, cuticular synthesis compensated fruit growth maintaining the weight per surface area. Total amount of polysaccharides diminished a 27% between 45 and 50 daa and then remained constant until red ripe. This decrease of polysaccharides coincided with the initiation of ripening (Brummell 2006) and had an effect in the amount of cuticle that also diminished in this period. The amount of cutin remained almost constant.



The amount of total waxes increased more slowly compared with the other cuticle components (Fig. 3B). At 25 daa, a halt in wax accumulation was observed accompanied by a decrease in their amount at 30 daa as the fruit expanded. Later on, the amount of waxes increased to the levels previously observed to, again, decrease from the breaker stage until red ripe. The epicuticular and intracuticular waxes mimicked the behaviour of the total waxes with a few exceptions (Fig. 3B). In the first 15 daa, the intracuticular waxes increased more rapidly than the epicuticular ones. From 15 to 20 daa, however, there was a 3-fold increase of epicuticular waxes, not accompanied by a similar boost of intracuticular waxes. From this period on, the ratio of epi- v. intracuticular waxes remained constant. At the red ripe stage, the

epicuticular waxes halved their amount and the intracuticular showed a slight increase. The amount of cuticle per fruit unit, calculated considering the fruit a sphere, increased during fruit growth until the fruit fully expanded at 40 daa (Fig. 4A), showing a positive correlation with fruit growth and cuticle thickness (Fig. 1B). Indeed, the maximum amount of cuticle per fruit coincided with the maximum cuticle thickness and fruit growth at 40 daa. This was also true for the cutin and polysaccharides components of the cuticle (Fig. 4A). After 40 daa, cutin decreased although a slight increase could be observed at the red ripe stage. Polysaccharides remained constant until 45 daa and then sharply decreased. The amount of waxes per fruit unit

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Days after anthesis Fig. 4. Evolution throughout development of the amount of micrograms per tomato fruit of (A) Cascada isolated cuticle and their components cutin and polysaccharides and (B) Cascada epicuticular, intracuticular and total waxes. Both graphs include the evolution of fruit diameter (mm) during growth. Data are presented as means s.e. of 20 measurements per developmental stage. The amount of micrograms per fruit was calculated considering the fruit a perfect sphere.


(Fig. 4B) showed a trend similar to the amount of waxes per surface area (Fig. 3B), the bimodal behaviour already mentioned. In general terms, two tendencies can be observed in the fruit and cuticle parameters measured in the present work. One was a fast increase in the first 15 days of tomato fruit development and the second a general reduction as the fruit ripened. In 2007, analyses of the amount of micrograms of cuticle per surface area at three critical stages, 15 dda, breaker and red ripe, were repeated with Cascada, Cornell Inbred 10, Gardener’s Delight and RIL115. The results confirmed 2006 data, reporting that at around 15 daa the fruit cuticle almost reached the maximum amount of micrograms per square centimeter. Thus, Cascada showed 1.21 and 1.22 mg cm--2 at 15 daa and red ripe (55 daa), respectively, Cornell Inbred 10 1.31 and 1.59, Gardener’s Delight 1.10 and 1.11 and RIL115 1.14 and 1.27 mg cm--2 at 15 daa and red ripe (45 daa), respectively. Likewise, 2007 results supported the general decrease in the amount of polysaccharides between breaker and red ripe. Again, Cascada showed a decrease in micrograms per square centimeter from 520.4 to 440.9 between breaker (45 daa) and red ripe (55 daa), Cornell Inbred 10 from 480.1 to 430.2, Gardener’s Delight from 407.5 to 340.3 and RIL115 from 454.5 to 399.9 between breaker (35 daa) and red ripe (45 daa). Discussion Fruit development in most higher plants can be divided into three phases (Gillaspy et al. 1993). The first is the development of the ovary and fruit set, the decision whether the ovary will abort or produce a fruit. In the second phase fruit growth is mainly due to cell division. In tomato, this event takes place for seven to 10 days after fruit set (Mapelli et al. 1978; Varga and Bruinsma 1986). The length of this phase is important since it has been observed in melon that the final size of the fruit will mostly depend on its duration (Higashi et al. 1999). The third phase begins after cell division ceases and fruit growth continues mostly by cell expansion until the fruit reaches its final size, usually 6--7 weeks after fruit setting, depending on the final size of the fruit. This agrees well with our observations: an increase in cell number can be observed from anthesis to 5 daa (Fig. 2A, B), and in the period between 5 and 10 daa cell enlargement starts in the parenchymal tissue (see Fig. 2C). Despite this, cell division still can be observed in the epidermal and hypodermal tissues (see Fig. 2D--F, I) at the beginning of this last phase, agreeing with the existing literature (Gillaspy et al. 1993). The initial lag phase in fruit growth, previously described in tomatoes (Ho and Hewitt 1986) and other fruits such as cherry (Peschel et al. 2007) seems to be absent or much reduced. This could be a characteristic of cherry tomatoes and, may be an effect of the short period of cell division. A similar timing can be observed between cuticle thickness, fruit growth and amount of cuticle per fruit unit in Cascada; but the amount of cuticle per surface area shows a very different pattern of growth with two remarkable points. One is the fast synthesis of cuticle in the early stages of fruit development and the second is the beginning of polysaccharide lost in the period from breaker to red ripe. These two features were confirmed with the three other cultivars Gardener’s Delight, Cornell Inbred 10 and RIL115 and suggest time frames where genetic studies regarding cuticle


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synthesis and/or polysaccharide degradation should be addressed. No significant differences in fruit and cuticle development were observed among the four genotypes that could be related to their sensitivity to cracking. This implies that the events leading to cuticle changes that affect cracking sensitivity cannot be traced back to fruit development but probably happen during fruit ripening. It is puzzling the fact that the cuticle expands its thickness as the fruit grows (Fig. 1B) while retaining the same amount of micrograms per square centimeter (Fig. 3A). It seems to be a paradox that could be explained by the putative accumulation of microscopical and/or nanoscopical cavities in the cuticle during fruit growth (Luque and Heredia 1994). This way, the void space could account for the increase in thickness without incrementing the final weight per surface area. This hypothesis deserves a further detailed study. One could also think of the enzymatic procedure used to isolate the cuticle as another possible explanation for this paradox since it might alter the polysaccharide content of the cuticle. This is not the case because cuticle thickness remains almost the same regardless it is measured from fresh epidermal tissue or isolated cuticle (López-Casado 2006). There are only two known studies on tomato cuticle composition during fruit growth (Baker et al. 1982; Luque and Heredia 1994). From their results, a model of slow cuticle accumulation during fruit growth was developed. Such model clearly contradicts our results that suggest a maximum accumulation in a very short period of time early in development followed by a steady increase in cuticle material that maintains this amount of cuticle per unit of fruit area. One possible explanation for the differences could be the fact that we studied cherry tomatoes while Baker et al. (1982) and Luque and Heredia (1994) used medium and big sized tomatoes in their research. A closer examination of our data and those by Baker et al. (1982) and Luque and Heredia (1994) suggest that, more than different models of cuticle growth, cherry tomato and medium-big sized tomatoes show different rates of cuticle synthesis. At 15 daa, when the fruit had reached 58.6% of the final volume, the amount of cuticle was 98.1% of the total observed at red ripe. In Baker et al. (1982) and Luque and Heredia (1994) a similar percentage of cuticle (95.2 and 91.7%, respectively) was achieved when the fruit had reached full expansion, at the mature green stage. In cherry tomatoes, the time from fruit set to ripe is shorter than in medium/big sized tomatoes, therefore, it may be that cuticle synthesis, as well as tomato growth and ripening, is sped in this type of tomatoes. Thus, two models of cuticle synthesis related to fruit development could be drawn: one of slow accumulation of cuticle material as the fruit expands for medium/big tomatoes, and another one of fast accumulation in the first stages of development for cherry tomato. This may have important physiological implications for the pattern of cuticle growth compared with that observed in normal tomato. The plant cuticle is a very complex polymer and little research has been focussed on the developmental evolution of physiological and biophysical parameters. One example is the observed increase in water permeability as fruit and cuticle grows in normal tomato (Luque and Heredia 1994). Therefore, parameters such as fruit dehydration, cuticle permeability, hydration degree and biomechanics must be studied during

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fruit growth and development in both types of tomato in order to establish a functional implication of the different patterns of cuticle growth. Recently, Peschel et al. (2007), working with cherry fruits, observed another pattern of fruit cuticle growth. In this case, there was a fast increase in the amount of cuticle in the first stages of development, similar to what we observed with cherry tomato, followed by a cease in cuticle synthesis during the rest of the growing period that rendered a less consistent cuticle at maturity. The percentage of each cuticle components, cutin, polysaccharides and waxes, was maintained throughout all fruit development, suggesting a very similar stechiometry of the cuticular components during all the stages. This is in disagreement with that observed by Baker et al. (1982) and Luque and Heredia (1994), where the ratio of polysaccharides decreased while the amount of cutin increased until a maximum was reached at red ripe. This clearly indicates a change in cuticle composition and most probably differences in mechanical and other physical properties throughout fruit development. The observed 2 : 1 ratio of intra: epicuticular waxes agrees well with the ratio recently reported by Leide et al. (2007) for tomato fruit cuticle from different cultivars. Another reason that could explain a faster or slower accumulation of cuticle was observed in the present work. In the experimental study of 2007, Cascada fruits at 15 daa were harvested earlier in the year (late April) than in 2006 (early June). We note that the 15 daa Cascada cuticles from 2007 exhibited a much lower amount of micrograms per surface area (805.2 mg cm--2) than the 15 daa Cascada from 2006 (1307.35 mg cm--2, Fig. 3A). A new set of Cascada fruits were harvested in May 2007 and the micrograms per surface area corroborated the previous results of 2006. A possible explanation could be the temperature, light intensity and/or relative humidity differences during the last half of April and May (Table 1). The comparison of the temperature, relative humidity and photosynthetic active radiation in the greenhouse during the 15-day period of fruit set and growth of the Cascada fruits harvested in April (maximum 24C, 94%; minimum 12C, 54%; mean 18C, 74%) and in May (maximum 29C, 95%; minimum 13C, 51%; mean 21C, 73%) showed an increase in temperature and photosynthetic active radiation (23--32 mol m 2 day 1) but almost no differences in relative humidity. The effects of temperature and light in fruit growth are known, but both sets of Cascada fruits at 15 daa exhibited similar diameters. The role of light in the accumulation of waxes has also been studied (Bringe et al. 2006), but no role has been assigned to temperature or light on the synthesis of the cuticle, either to the cutin matrix or the polysaccharides. Together, these preliminary data suggest a role for the environmental conditions in the rate of cuticle synthesis in a growing fruit that should deserve a careful study. Acknowledgements The authors thank Dr R. Fernández-Muñoz for critical reading the manuscript and Ana Rico for technical assistance. This work has been partially supported by grant AGL2006--12494 from Plan Nacional de I+D, Ministerio de Educación y Ciencia, Spain, Fundación Cajamar and Rijk Zwaan Iberica (Almería, Spain).


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Manuscript received 25 January 2008, accepted 6 May 2008

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