Accumulation of health promoting phytochemicals in wild relatives of tomato and their contribution to in vitro antioxidant activity

Phytochemistry 71 (2010) 1104–1114

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Accumulation of health promoting phytochemicals in wild relatives of tomato and their contribution to in vitro antioxidant activity Antonio J. Meléndez-Martínez 1, Paul D. Fraser, Peter M. Bramley * School of Biological Sciences, Royal Holloway, University of London, Egham TW20 0EX, UK

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Article history: Received 23 November 2009 Received in revised form 29 March 2010 Available online 8 May 2010 Keywords: Antioxidants Biosynthesis Carotenoids Carotenogenic genes Colour Gene expression Phenolics Phytochemicals Phytoene synthase (Psy-1) Tocopherols Tomato wild relatives

a b s t r a c t Harnessing natural variation is an important aspect of modern marker assisted breeding. Traditionally breeding programmes have focused on increased yield and resistance to biotic and abiotic pressures. However, consumer demands for improved quality have lead to increased effort into the breeding of nutritional quality traits in crop plants. In the present study, health-related phytochemicals (carotenoids, tocopherols and phenolics) present in green, yellow and red wild relatives of tomato have been analyzed during fruit development and ripening. This study shows that the differences in the final colour of the fruits were due to a distinct accumulation of carotenoids mainly related to the expression of the phytoene synthase-1 gene (Psy-1). In ripe red-fruited tomatoes, the different deposition of pigments gave rise in some cases to colour differences visually discernible by the consumer. Important quantitative differences between and across taxa were noticed for the in vitro antioxidant activity (AA) of the samples. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Diets rich in fruits and vegetables are being encouraged due to their inverse relationship with the incidence of chronic diseases (Steinmetz and Potter, 1996; Southon, 2000). This beneficial effect is thought to be related to the presence of diverse secondary metabolites, which are commonly referred to as phytochemicals. The tomato and its products are being extensively studied in connection with this dietary advice, since they are food staples and contain diverse health-related antioxidants such as carotenoids, tocopherols and flavonoids (Frusciante et al., 2007; Abushita et al., 1997; Caris-Veyrat et al., 2004; Seybold et al., 2004; Toor and Savage, 2005). Carotenoids are a group of ubiquitous pigments found in plants, algae, fungi, bacteria and animals. They account for the colours of many plants and animals (De Rosso and Mercadante, 2005; Englberger et al., 2003; Konopka et al., 2004; Tadmor et al., 2005; Cremades et al., 2003; Ronsholdt and McLean, 2001), are precursors of aroma compounds (Baldermann et al., 2005; Silva Ferreira et al., 2008) and play essential roles in different processes in plants. They

* Corresponding author. Tel.: +44 (0)1784 443555; fax: +44 (0)1784 434326. E-mail address: [email protected] (P.M. Bramley). 1 Present address: Laboratory of Food Colour and Quality, Department of Nutrition and Food Science, Faculty of Pharmacy, Universidad de Sevilla, 41012 Sevilla, Spain. 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.03.021

are also essential components of the photosynthetic reaction complexes. In addition, many carotenoids are potent lipophilic antioxidants and are thought to prevent the onset of chronic disease states (Krinsky, 1989; Nishino, 1998; Snodderly, 1995; Carrapeiro et al., 2007). The biosynthesis of carotenoids in plants is well characterized at the biochemical and molecular levels (Fig. 1), although the understanding of its regulation remains poor, despite recent advances in the field. In this regard, several transgenic lines of tomatoes and other foods from plants have been developed, although their acceptability by consumers remains an issue (reviewed in Fraser and Bramley (2004)). Tocopherols have been reported to be involved in several plant processes (DellaPenna and Pogson, 2006; Maeda and DellaPenna, 2007). Nutritionally, the tocopherols (a-, b-, c- and d-tocopherol) and tocotrienols are important because they exhibit vitamin E activity. Additionally, they have been traditionally regarded as potent physiological antioxidants (Rock et al., 1996), although their in vivo mechanism of action remains little understood (Azzi, 2007; Traber and Atkinson, 2007). In higher plants, carotenoids and other biosynthetically-related isoprenoids (tocopherols, gibberellins, chlorophylls, and phylloquinones) are synthesized in plastids from the five-carbon (C5) precursor isopentenyl diphosphate (IPP). Specifically, the immediate precursor of the carotenoid pigments is the 20-carbon compound geranylgeranyl pyrophosphate (GGPP), formed from four C5

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likely beneficial effect on the prevention of cancer and other serious human disorders (Hollman et al., 1996; Le Marchand, 2002; Tripoli et al., 2007). Biosynthetically, the majority of flavonoids derive from malonyl-CoA and p-coumaroyl-CoA. The biosynthesis of flavonoids commences with the synthesis of chalcone, which is catalyzed by the enzyme chalcone synthase (CHS), although depending on the plant, the final products are diverse (reviewed by Schijlen et al. (2004)). In the case of tomato, the major flavonoids are typically rutin (quercetin-3-rutinoside) and naringenin chalcone, with the levels of another phenolics, chlorogenic acid, being noteworthy (Long et al., 2006). In this study we have studied the changes in the phenolics and isoprenoid profiles, derived in vitro antioxidant activity and colour of 10 wild relatives of tomato during fruit ripening in the context of the new trends related to the molecular breeding of wild relatives into backgrounds to shed light in the understanding of their biodiversity. Our main objective was to build upon previous studies devoted to the understanding of the mechanisms that govern the biosynthesis of carotenoid pigments and other phytochemicals in diverse tomato lines (Davuluri et al., 2005; Fraser et al., 1994, 2001, 2002; Long et al. 2006; Römer et al. 2000). The study of this collection of ancestors of the domesticated tomato is particularly interesting for this purpose since it includes genotypes producing stay-green, yellowish and red tomatoes. 2. Results 2.1. Changes during fruit development and ripening of chlorophyll, carotenoid and tocopherol

Fig. 1. Summary of biosynthetic steps involved in the formation of diverse isoprenoids. GGPP: geranylgeranyl diphosphate; PSY: phytoene synthase; CYC-B lycopene cyclase B.

isoprene units, which condense with a further molecule of GGPP to form the C40 carotene phytoene (7,8,11,12,70 ,80 ,110 ,120 -octahydrow,w-carotene). This first biosynthetic step specific of carotenoids is catalyzed by phytoene synthase, which in the case of tomato is synthesized in chloroplasts by the Psy-2 gene product and in chromoplasts by the Psy-1 gene product. Once phytoene (three conjugated double bonds) is synthesized, a series of desaturation reactions takes place sequentially and lycopene (11 conjugated double bonds) is eventually formed. This carotenoid is the immediate precursor of cyclic carotenoids, which in plants are primarily synthesized by two major cyclases, namely lycopene-b-cyclase and lycopene-e-cyclase (LCY-b and LCY-e, respectively). In the case of tomatoes two Lcy-b genes have been identified, one of them (Cyc-b) exhibiting chromoplast-specific expression. From this point onwards, diverse hydroxylation and epoxidation reactions can take place to yield a wide array of xanthophylls (Fig. 1) (reviewed in Fraser and Bramley (2004)). Phenolics (flavonoids, tannins, phenolic acids, coumarins, stilbenes, xanthones, anthraquinones, etc.) are one of the main groups of plant secondary metabolites, alongside terpenoids and alkaloids. Chemically, the flavonoids (anthocyanins, chalcones, flavones, isoflavones, etc.) consists of two benzene rings linked by an oxygenated heterocyclic ring and they can occur as glycosides containing a sugar moiety (rutinose, rhamnose, glucose, galactose, and arabinose), or as aglycones (without the sugar moiety). They serve important functions in plants (Li et al., 1993; Harborne and Williams, 2000) and are increasingly attracting attention for their antioxidant, antiinflammatory and antimicrobial properties, and their

2.1.1. Chlorophyll pigments The levels of chlorophyll pigments at immature green (IG, 11 ± 2 days after anthesis) stage in red-fruited accessions differed significantly (Fig. 2). The content in accession 1511 (Solanum lycopersicum var. cerasiforme), 632.87 lg/g of dry matter, was some 4fold higher than that of accession 0114C (S. pimpinellifolium), 1.6fold higher than accession 0114C (S. lycopersicum) and 1.4-fold higher to that of the other S. lycopersicum var. cerasiforme genotype (accession 2675). In the remaining accessions the total chlorophyll content decreased from ST1 to ST3 (Fig. 3), except for the case of accession 1984 (S. peruvianum). The highest chlorophyll content (437.25 lg/g dw) was found in ST1 accession 2744 (S. peruvianum) fruits, which was some 1.7-fold higher than that of the next accession with the ST1 fruits with highest pigmentation (accession 2695, S. chmielewskii) and 3.8 higher to that of the least pigmented ST1 fruits (accesion 2879, S. chilense). The ratios chlorophyll a + pheophytin a to chlorophyll b at ST1 stage ranged from 1.63 (accession 2744, S. peruvianum) to 2.08 (accession 2879, S. chilense). 2.1.2. Carotenoid pigments The changes in the levels of the carotenoid pigments over the development and ripening of red-fruited accession are depicted in Fig. 2. The main coloured carotenoids in the chloroplast-containing IM and mature green (MG, 37 ± 1 days after anthesis) stages were lutein and b-carotene, respectively. The levels of the other carotenoids in chloroplast-containing tissues, namely violaxanthin and (90 Z)-neoxanthin were negligible. The colourless carotenoids phytoene and phytofluene (7,8,11,12,70 ,80 -hexahydro-w,w-carotene) were not detected in these stages. In relation to the individual changes in the levels of carotenoids from the breaker (BR, 44 ± 1 days after anthesis) through the ripe (RI, 10 days after breaker) stage, it was seen that while the amount of b-carotene increased steadily in all the genotypes, the eventual reduction in the levels of lutein did not follow the same pattern in all of them

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0114 (S. pimpinellifolium) dotted bar 0134C (S. lycopersicum) striped bar 1511 (S. lycopersicum var. cerasiforme) black bar 2675 (S. lycopersicum var. cerasiforme) white bar Fig. 2. Changes in the levels of isoprenoids (lg/g dw) over development and ripening in red-fruited wild relatives (three fruits per stage for S. lycopersicum and >20 for the rest, three analytical replicates).

(Fig. 2). Lutein content diminished steadily in all the accessions except for 1511 (S. lycopersicum var. cerasiforme), where a striking 1.4-fold rise in the levels of lutein from the breaker to the turning

(TU, 3 days after breaker) stage was observed. The highest levels of b-carotene (151.44 lg/g dw, accession 2675, S. lycopersicum var. cerasiforme) in the ripe stages were lower than those of Ailsa Craig

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tomatoes (271.0 lg/g dw) and transgenic tomatoes transformed with the CrtI bacterial gene (520.0 lg/g dw) analyzed in our laboratory previously (Römer et al., 2000). The highest amounts of lycopene in ripe fruits (1985.93 lg/g dw, accession 2675, S. lycopersicum var. cerasiforme) were lower than those found in Ailsa Craig tomatoes (2436.0 lg/g dw), but higher relative to those of transgenic tomatoes transformed with CrtI (733.0 lg/g dw) analyzed in a previous study (Römer et al., 2000). Concerning the colourless carotenoids, phytofluene was detected at the TU (turning) and RI (ripe) stages, although their levels were lower than those of phytoene. The latter was found to increase from the breaker (BR, 44 ± 1 days after anthesis) to the RI stages in all the red-fruited accessions. Its levels at the RI stage ranged from 65.18 lg/g dw in accession 1511 (S. lycopersicum var. cerasiforme) to 302.12 lg/g dw in accession 2675 (S. lycopersicum var. cerasiforme), and were typically 2–3-fold higher than those of b-carotene. To explain the deposition of carotenoids at the molecular level in these red-fruited tomatoes, the changes in the expression of Psy-1, Psy-2 and Cyc-B genes over a ripening series were studied in accession 0134C (S. lycopersicum). The expression of Psy-1 increased sharply from the mature green to the breaker (266-fold) stage and continued to increase from this stage to the ripe one (1.8-fold). The expression of Cyc-B remained negligible regardless of the ripening stage, whereas that of Psy-2 remained at low levels from MG to the ripe stage. In the green- and yellow-fruited accessions only lutein (60.22– 81.45% of the total carotenoid content) and b-carotene (11.45– 39.61% of the total carotenoid content) were detected. The changes in the levels of both pigments along the fruit development time course were not uniform across genotypes (Fig. 3). As far as lutein was concerned, its levels increased from ST1 to ST3 in the accessions 1937 (S. peruvianum, 1.4-fold), 1984 (S. peruvianum, 1.5-fold) and 2695 (S. chmielewskii, 1.75-fold), whilst they decreased in the

remaining S. peruvianum genotype (3.9-fold) and in the two S. chilense accessions studied (1.1- and 2.3-fold, for accessions 2879 and 2759). Conversely, decreases in the levels of b-carotene ranging between 1.1-fold (accession 2879, S. chilense) and 11.5-fold (2744, S. peruvianum) were observed in all the accessions save for accession 1984 (S. peruvianum), were the amount of such carotene increased some 3-fold. To gain an insight into the molecular basis of the carotenoid deposition in the stay-green accessions, the expression of pertinent carotenogenic genes during ripening was evaluated in accession 1984 (S. peruvianum). The expression of Psy-1 and Psy-2 increased from ST1 to ST3 (46- and 15-fold, respectively). The expression of Psy-2 was some 7-fold lower as compared to that found in the ripe stages of accession 0134C, although that of Psy1 was a staggering 2364-fold lower. The expression of the Cyc-B gene in accession 1984 increased from ST1 to ST2 to remain much constant at ST3. Its expression in the stay-green tomato was higher relative to that in the red-fruited one, regardless of the maturity stage considered. Since phytoene synthase 1 is the enzyme that exerts the highest control over the carotenoid pathway flux (Fraser et al., 2002), the expression of Psy-1 was also studied across accessions at the ripe (red-fruited accessions) or ST3 (remaining accessions) stages. The results are shown in Fig. 4, where it can be observed that the expression of this gene in the green- and yellow-fruited tomatoes was negligible regardless of the accession considered. When the relationship between the expression of the Psy-1 gene and the total carotenoid content of such stages was explored by regression analysis, an r value of 0.78 was obtained. 2.1.3. Tocopherols In addition to a-tocopherol, detectable levels of c-tocopherol were found in almost all the red-fruited accessions at all the stages. The levels of a-tocopherol varied from 32.62 lg/g dw (IG stage of

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accession 0114, S. pimpinellifolium) to 178.21 (TU stage of accession 1511, S. lycopersicum var. cerasiforme) and those of c-tocopherol from not detected (MG stage of accession 1511, S. lycopersicum var. cerasiforme) to 87.35 lg/g dw (TU stage of accession 2675, S. lycopersicum var. cerasiforme). The tocopherol levels were higher than those reported for ripe Ailsa Craig and Cnr mutant tomatoes (6.0 and 7.3 lg/g dw, respectively) (Fraser et al., 2001). In general, the levels of c-tocopherol increased from the IG through the TU stages, after which they dropped. In the case of a-tocopherol, a slight reduction in its levels was observed from the IG to the MG stage in three of the four accessions, after which they increased at the breaker stage and remained quite constant up to the ripe stage (Fig. 2). The ratio of a-tocopherol to c-tocopherol either increased slightly (some 1.1-fold in the case of accessions 0114 and 2675) or dropped (around 3-fold in the case of accessions 0134C and 1511) from the IG to the RI stages. Concerning the relationships with the other isoprenoids included in this study, it was noticed that the ratio of total chlorophylls to total tocopherols ranged between 3.78 (accession 0134C, S. esculentum) and 7.41 (accession 1511, S. esculentum var. cerasiforme) at the IG stage and diminished steadily until the depletion of the chlorophyll pigments. However, the total carotenoids to total tocopherols ratio diminished slightly (1.22–1.59-fold) from the IG to the MG stage, to later increase steadily until the TU stage and more sharply at the RI stage, the total increases from IG to R stages ranging from 6.36-fold (accession 1511, S. esculentum var. cerasiforme) to 35-fold (accession 0114, S. pimpinellifolium). In the case of the green- and yellow-fruited accessions, only atocopherol was detected in the accessions 1937 and 2744 (S. peruvianum). In the former accession its level increased 1–82-fold from ST1 to ST3 and in the latter decreased 1.22-fold, which indicated clear varietal differences in its biosynthesis. 2.2. Changes occurring over fruit development and ripening of phenylpropanoids and flavonoids The main phenolic compounds in red-fruited accessions were the flavonoids rutin (quercetin-3-O-rutinoside) and chalcone naringenin, which are known to accumulate in the peel (Muir et al., 2001), as well as the hydroxycinnamic acid derivative chlorogenic acid. In the remaining accessions, either rutin or chlorogenic acid were typically the major phenolic compounds, chalcone naringenin was not detected and some unknown compounds not present in red-fruited accessions were detected occasionally. In the red-fruited accessions the accumulation of rutin along the ripening of the fruits did not follow a definite pattern across accessions (Fig. 5). In the case of accession 0134C there was an eventual 2.4-increase in its levels from the IG to the R stage, whilst in the remaining accessions the amount of that flavonoid decreased between 1.12- and 1.92-fold. The levels of chlorogenic acid decreased sharply from the IG to the R stage (e.g., between 1.52-

and 4.90-fold) in all the accessions. Chalcone naringenin, however, was detected from the breaker stage onwards, and its levels increased from the BR to the T stage to decrease eventually (Fig. 5). The highest contents of rutin (1262 lg/g dw) and chlorogenic acid (3139.63 lg/g dw) were found at the breaker stage of accession 2675 (S. lycopersicum var. cerasiforme), and those of chalcone naringenin (1436.60 lg/g dw) at the turning stage of accession 2675 (S. lycopersicum var. cerasiforme). In the accessions other than the red-fruited it was noticed that rutin remained virtually the same in accessions 1984 and 1937 (S. peruvianum), diminished some 1.6-fold in accession 2695 (S. chmielewskii) and disappeared in accession 2744 (S. peruvianum) and 2759 (S. chilense). In the case of chlorogenic acid, a clear decrease in its levels was observed in all the accessions, specifically to depletion in the case of accession 2695 (S. chmielewskii), and between 1.6-fold (accession 1.63, S. peruvianum) and 9.2-fold (accession 2759, S. chilense) in the remainder. The highest levels of rutin (1341.97 lg/g dw) and chlorogenic acid (6457.27 lg/g dw) were found in the stage 1 of accession 1984 (S. peruvianum) and the stage 1 of accession 2695 (S. chmielewskii). Comparing the levels of phenolics reported in this study with those reported for the domesticated tomato cultivar Ailsa Craig (Long et al., 2006) (200–1300 lg/g dw of chlorogenic acid, 3500– 6000 lg/g dw rutin and 2500–6000 lg/g dw for chalcone naringenin) it was observed that the levels of rutin and chalcone naringenin in this domesticated variety were higher than the highest amounts found in the wild relatives studied, which was not the case for chlorogenic acid. 2.3. Objective colour assessment In the case of red-fruited tomatoes it was observed that immature green and mature green tomatoes showed a very similar colour. Considering the coordinates a* (that takes positive values for reddish colours and negative values for greenish ones) and b* (that is positive for yellowish colours and negative for bluish colours) it was seen that they exhibited negative values of a* (ranging from 7.12 in the IG stage of accession 1511 S. lycopersicum var. cerasiforme to 3.41 in the MG stage of accession 0114 S. pimpinellifolium) and positive values of b* (ranging from 11.24 in the MG stage of accession 0114 S. pimpinellifolium to 23.37 in the IG stage of accession 1511 S. lycopersicum var. cerasiforme). Their hue values (hab, the qualitative attribute of colour) varied from 104.88° (MG stage of accession 1511 S. lycopersicum var. cerasiforme) to 108.74° (IG stage of accession 2675 S. lycopersicum var. cerasiforme) and their chroma (C ab , the quantitative attribute of colour) from 11.75 (MG stage of accession 0114 S. pimpinellifolium) to 24.42 (IG stage of accession 1511 S. lycopersicum var. cerasiforme). From these stages onwards, a progressive increase in the values of a* and a decrease in the values of the hue angle were observed. The most dramatic change in a* from the IG to the ripe stage (from 7.12 to 19.16, an increase of roughly 26 CIELAB units) was observed in accession 1511 (S. lycopersicum var. cerasiforme), while the sharpest decrease in the hab (from 108.74° to 38.18°, around 70.5° in difference) was observed in accession 2675 (S. lycopersicum var. cerasiforme). As for b* and C ab , it was observed that, in general, values increased from the green stages to the turning one to decrease eventually, although this final decrease was clearly lower in the case of the chroma. All these changes in the colour coordinates were accompanied by a concomitant decrease in L* (approximate measure of lightness), approximately from 50 to 30 units. Considering the quantitative attribute of colour, i.e., chroma, it was seen that the ripe tomatoes with the highest (27.30 CIELAB units) and lowest values (20.37 CIELAB units) were accession 1511 (S. lycopersicum var. cerasiforme) and accession 0114 (S. pim-

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0114 (S. pimpinellifolium) dotted bar 0134C (S. lycopersicum) striped bar 1511 (S. lycopersicum var. cerasiforme) black bar 2675 (S. lycopersicum var. cerasiforme) white bar Fig. 5. Changes in the levels (lg/g dw) of rutin, chalcone naringenin and chlorogenic acid over development and ripening in red-fruited wild relatives (three fruits per stage for S. lycopersicum and >20 for the rest, three analytical replicates).

pinellifolium), respectively. Regarding the relationship between the expression of the Psy-1 gene across accessions in ripe (red-fruited accessions) or ST3 (stay-green accessions) tomatoes and their colour, it was seen that gene expression was highly correlated to a*, hab, L* and C ab (r = 0.93, 0.88, 0.85 and 0.82, respectively) and correlated to a much lesser extent to b* (r = 0.46). 2.4. In vitro antioxidant activity The red-fruited accessions with the lowest (accession 2675, 40.55 mmol/kg dw) and highest total antioxidant activity (TAA) (accession 1511, 126.78 mmol/kg dw) belonged to the S. lycopersicum var. cerasiforme taxa, and exhibited an approximately 3-fold difference in their in vitro antioxidant activity. The highest antioxidant activity in methanol extracts (AAMeOH) (52.84 mmol/kg dw) was found at the IG stage of accession 0134C (S. lycopersicum) and the lowest (17.76 mmol/kg dw) at the IG stage of accession 2675 (S. lycopersicum var. cerasiforme). In accessions 0114 and 2675 (S. pimpinellifolium and S. lycopersicum var. cerasiforme, respectively) the AAMeOH remained almost constant, whilst in accessions 0134C and 1511 (S. lycopersicum and S. lycopersicum var. cerasiforme, respectively), a clear drop from the IG to the ripe stage was noticed. The accessions with the lowest and highest antioxidant activity in the ethyl acetate extract (AAEtAc) when ripe were accession 2675 (S. lycopersicum var. cerasiforme, 20.79 mmol/kg dw) and accession 1511 (S. lycopersicum var. cerasiforme,

99.24 mmol/kg dw), which displayed a dramatic difference (roughly 5-fold) in their AAEtAc. The accessions other than the red-fruited accessions with the lowest and highest TAA at ST3 were accession 1937 (S. peruvianum, 43.24 mmol/kg dw) and accession 2879 (S. chilense, 97.61 mmol/kg dw). The AAMeOH found at the ST3 stages of accession 1984 (S. peruvianum), 2759 and 2879 (S. chilense) (67.99, 82.20 and 82.96 mmol/kg dw, respectively) were much higher than those found in the remaining stay-green and red-fruited accessions. With respect to the changes of AAMeOH over ripening, it increased smoothly in accessions 1937, 1984 (S. peruvianum) and 2695 (S. chmielewskii) and sharply in accessions 2759, 2879 (S. chilense) and, above all, 2744 (S. peruvianum) (Table 2). The AAEtAc values in all the stages of the stay-green accessions were normally much higher relative to the green stages (IG and MG) of the red-fruited ones. The highest AAEtAc (22.29 mmol/kg dw) was found at the ST3 stage of accession 1937 (S. peruvianum), and the lowest (10.70 mmol/kg dw) at the ST3 stage of accession 2695 (S. chmielewskii). 3. Discussion The deposition of phytochemicals in ten wild relative of tomatoes has been studied in the context of the trends towards the exploitation of wild genetic resources. The harnessing of these resources is beneficial because they can improve the nutritional or

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economical value of crops and provide the molecular basis of their adaptation to different habitats, in addition to favour the genetic diversity of the family. The wild relatives analyzed in this study encompassed plants bearing red, green and yellow ripe fruits and were chosen to study the accumulation of isoprenoids and phenolics in related species. 3.1. Chlorophyll, carotenoid and tocopherol contents and changes over fruit development and ripening In red-fruited accessions, chlorophylls a, b and pheophytin a were found in the early stages (immature green through breaker) of all the genotypes. Their levels diminished progressively as is the norm in the ripening process of these products. The presence of pheophytin a arises from the occurrence of pheophytinization, which is favoured by senescence and the disruption of cell structures during the handling of the samples. However, no detectable levels of pheophytin b were found. In the remaining accessions, chlorophyll a, chlorophyll b and pheophytin a, but not pheophytin b were found at the three stages of maturity considered as expected. In all the accessions studied the ratio of chlorophyll a to chlorophyll b increased along the chloroplast-containing stages considered, which may indicate that the rate of deterioration of the latter chlorophyll is higher. In red-fruited tomato genotypes the total content of carotenoids increased noticeably from the breaker to the ripe stage due to the transition of chloroplasts into chromoplasts. More specifically, high amounts of lycopene were synthesized and sequestrated in the form of crystals as a result of increased phytoene synthesis conferred by transcriptional up- and concurrent down-regulation of carotene cyclization into cyclic carotenes and their downstream oxygenated derivatives (reviewed in Fraser and Bramley (2004)). The amounts of lutein and b-carotene were reduced from the IM to the MG stage at virtually the same rate as were the chlorophyll pigments, as they form part of the photosynthetic machinery (Demmig-Adams et al., 1996). Thus, the accession with the highest and lowest carotenoid contents at IM stage coincided with those with the extreme contents in chlorophylls. These facts add evidence to another report (vonLintig et al., 1997) indicating that the expression of phytoene synthase (in this case Psy-2) forms part of an orchestrated series of events related to the operation of the photosynthetic apparatus. With the onset of the breaker stage, important changes in the structure of the chloroplasts and in the expression of genes involved in the metabolism of carotenoids are initiated. The thylakoids swell and, as a consequence of the degradation of their lipids, lipid droplets and plastoglobuli appear, which contain free and/or esterified carotenoids and fatty acids, This senescence process is paralleled by a reduction in the levels of starch (reviewed in Heaton and Marangoni (1996)). Increases in the expression of phytoene synthase 1 and phytoene desaturase and depletion of the mRNA of both lycopene cyclases have been described (reviewed in Fraser and Bramley (2004)). Such changes lead to a progressive accumulation of high amounts of lycopene. Thus, it has been reported that in ripe tomato fruits lycopene accounts for well over 50% of the total carotenoid content, and the remaining major carotenoids are phytoene, lutein and b-carotene (Abushita et al., 2000; Burns et al., 2003). As a result of the dismantling of the photosynthetic apparatus and the changes in the expression of carotenogenic genes, the b-carotene/lutein ratio (ranging between 0.37 and 0.69 in IG and MG stages) clearly inverts. The values for such ratio in the breaker stages of the wild relatives analyzed were around or over 1.5, so this inversion can be used as a useful objective index to assess more precisely the onset of the breaker stage of tomatoes. Moreover, it was noted that the lutein content decreased from the breaker to the ripe stage in all the accessions except 1511 (S. lycopersicum var. cerasiforme),

which may be indicative of an unusual up-regulation of b- and ehydroxylases cyclases during the fruit ripening in this case. Considering the levels of carotenoids in accessions other than the red-fruited it can be inferred that important differences in the regulation of their biosynthesis did take place both within and between species. From the transcript analyses it can be concluded that the fact that the stay-green accessions that do not accumulate the typical carotenoids found in ripe red-fruited tomatoes is not due to the lack of the key carotenogenic genes studied, but to their low expression. The absence of such carotenoids may be brought about by the alteration of concomitant fruit ripening processes, such as softening or ethylene synthesis among others (Giovannoni, 2004; Alexander and Grierson, 2002), or by other factors, as it has been reported that fruit phytochromes are involved in the synthesis of lycopene (Alba et al., 2000). In relation to the role of ethylene in the maturation of tomatoes, it has been recently reported that the hormone coordinates a high transcript accumulation in fruits, such that a simple mutation in one ethylene receptor (responsible of the Never-ripe [Nr] phenotype) affects the expression of over 300 genes. More specifically, it was concluded by transcriptome/metabolite analysis that ethylene is involved in several carotenoid biosynthesis processes affecting their qualitative and quantitative deposition (Alba et al., 2005). a-Tocopherol and c-tocopherol were found virtually at all the stages of the red-fruited accessions, whereas the former was only found very occasionally in the remaining genotypes studied. The study of changes in their levels indicated that there were important differences in the regulation of their biosynthesis both between and within species. 3.2. Changes occurring over fruit development and ripening of phenylpropanoids and flavonoids Important quantitative and qualitative differences in the phenolic profile of red-fruited accessions and the remainder were observed, which was indicative of differences in the biosynthetic steps leading to the formation and accumulation of these metabolites between genotypes. The enzymes involved in the biosynthesis of the phenolics are well known. Chalcone naringenin is an intermediate in the synthesis of flavonols formed in a reaction catalyzed by chalcone synthase (CHS), whereas the quercetin glycoside rutin (quercetin-3-O-rutinoside) requires of the enzyme flavonol synthase (FLS) and glycosyltransferases to be synthesized. Chlorogenic acid is also synthesized in the phenylpropanoid pathway from cinnamic acid with the intervention of glucosyltransferase enzymes (Weisshaar and Jenkins, 1998; Muir et al., 2001; Aksamit-Stachurska et al., 2008). The changes in the levels of rutin (except in the case of accession 0134C) and chlorogenic acid indicated that the enzymes involved in their synthesis were down-regulated during development and ripening. The pattern of accumulation of chalcone naringenin from the breaker stage onwards is consistent with the down-regulation of the fls gene. In the accessions other than the red-fruited ones, a down-regulation of the biosynthetic steps leading to the formation of chlorogenic acid was also observed, whereas the accumulation of rutin was differently regulated across the taxa. In the light of the results obtained it can be claimed that, in general, the levels of phenolic antioxidants decreased as development and ripening proceeded. In summary, the highest levels of chlorophylls and a-tocopherol were found in the IG and TU stages of accession 1511 (S. lycopersicum var. cerasiforme), although such accession also exhibited the lowest carotenoid contents in ripe fruits and the lowest content of c-tocopherol of all the accessions and stages in its MG fruits. The lowest contents of chlorophylls in IG fruits were found in accession 0114C (S. pimpinellifolium), which also exhibited the lowest content of a-tocopherol of all the accessions and stages consid-

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ered. Lastly, the highest contents of carotenoids and c-tocopherol were detected in ripe and turning fruits of accession 2675 (S. lycopersicum var. cerasiforme).

of Psy-1 in ripe red-fruited tomatoes can give rise to colour differences easily distinguishable visually by the consumers, which has potentially important implications for their acceptability.

3.3. Objective colour assessment

5. Experimental

The colour difference between the ripe red-fruited tomatoes with extreme chroma values was roughly 7.5 CIELAB units, well over 5.6 CIELAB units, which is considered for industrial purposes as the threshold over which colour differences can be readily detected by people with normal colour tolerances (Lozano, 1978; Melgosa et al., 2001; Meléndez-Martínez et al., 2004). The colour differences between accessions 0134C and 0114 (S. lycopersicum and S. pimpinellifolium, respectively), were also above that threshold, unlike those corresponding to the remaining possible pairs, differences in the colour of which could not be easily detected visually (Table S2). In relation to these findings, it is to be noted that the relative differences in the expression of Psy-1 in those accessions at the ripe stage are very different. This indicated that the expression of such gene does play a key role in the eventual colour of the ripe fruits, which is to be expected since PSY-1 is the enzyme exhibiting the highest control over the carotenoid pathway flux (Fraser et al., 2002).

5.1. Generation of plant material

3.4. In vitro antioxidant activity For each red-fruited wild relative, the highest total antioxidant activity (TAA) was found at the ripe stage. Considering the AA of the methanol extracts (AAMeOH) along the ripening process it was noticed that it was normally higher relative to that of the ethyl acetate extract (AAEtAc) (Table 2), except for the ripe stage where AAEtAc was much higher in all cases due to the high deposition of lycopene at that stage. The highest AAMeOH were normally found in either the IG or MG stages, although their changes over ripening did not follow a fixed pattern, which reflects somehow the nonuniform deposition of phenolic compounds. With regards to the AAEtAc, it was seen that it increased from the IG to the ripe stage in all cases (roughly between 3- and 20-fold), mostly due to the deposition of lycopene, although different patterns of variation across the ripening stages were observed (Table 2). The fact that the accessions with the lowest and highest AAEtAc when ripe were from the same taxa (S. lycopersicum var. cerasiforme) is noteworthy, and is a good illustration of the complexity of the regulation of the biosynthesis of carotenoids even within the same variety. In the case of the green- and yellow-fruited wild relatives, the highest values of in vitro TAA were normally found in ST3 tomatoes. Regarding AAMeOH, it was observed that its values were higher than AAEtAc in most of the cases, since these wild relatives did not accumulate lycopene (the majority did not accumulate tocopherols either) and the levels of b-carotene were lower as compared to the red-fruited tomatoes. Contrarily to what occurred in the redfruited tomatoes, the highest AAMeOH values were normally found at the end of the ripening process (ST3).

Ten wild accessions of tomato (S. lycopersicum L.) from the Andean region (Table 1) were selected. Five plants from each accession were cultivated under controlled glasshouse conditions with supplementary lightning. Those accessions yielding red fruits were harvested at five designated developmental stages immature green (IG, 11 ± 2 days after anthesis), mature green (MG, 37 ± 1 days after anthesis), breaker (BR, 44 ± 1 days after anthesis), turning (TU, 3 days after breaker) and ripe (RI, 10 days after breaker). Accessions not yielding red fruits were harvested at three stages designated ST1, ST2 and ST3 on the basis of their relative fruit size. Harvested fruits (three per plant in the case of accession LA0134C and 15 per plant in the others) were deseeded and the remaining tissue frozen immediately in liquid nitrogen and kept at 80 °C. RNA was extracted from this frozen material. The material for metabolite analysis was lyophilized and then ground to a fine powder using a SPEX 6750 freezer mill (SPEX SamplePrep Inc., Metuchen, New Jersey) set at 1 cycle and rate 10. This material was stored at 20 °C prior to analysis. 5.2. Extraction and HPLC analysis of isoprenoids (carotenoids and tocopherols) The analysis of isoprenoids was performed as described elsewhere (Fraser et al., 2000). In brief, to powdered freeze-dried tomato tissue (10 mg) 250 ll MeOH was added and then vortexed. To this suspension 500 ll of chloroform was added and the mixture vortexed again. After adding 250 ll of Tris–HCl (50 mM, pH 7.5, containing 1 mM NaCl) and mixing, a partition was formed by centrifugation at 3000 rpm. The lower phase formed was removed. The remaining aqueous phase was re-extracted with a further 500 ll of chloroform and the pooled chloroform extracts were dried under nitrogen. The dried residue was stored at 20 °C prior to analysis or re-dissolved in 100 ll of ethyl acetate and centrifuged at 3000 rpm to remove particulates. Aliquots (20 ll) of the supernatant were injected into the HPLC. In all cases, three biological analysis were performed. The HPLC analyses were performed on an Agilent 1100 series system comprising degasser, solvent delivery system, photodiode array detector and fluorescence detector (Agilent Technologies UK Ltd., Wokingham, United Kingdom). Separations were performed on a C30 reversed-phase column (250  4.6 mm, YMC Inc., Wilmington, NC, USA), coupled to a 20  4.6 mm C30 guard column (YMC Inc., Wilmington, NC, USA) and maintained at

Table 1 Wild relatives of tomato used in this study.

4. Conclusions

Accession

Seed lot

In summary, it can be concluded that there are important qualitative and quantitative differences in the formation and deposition of health-related phenolics and isoprenoids across the diverse genotypes studied, which is reflected in their relative in vitro antioxidant potency. With respect to carotenoids, the absence of the typical carotenoids found in red-fruited tomatoes in the other accessions was attributed to the low levels of transcripts from key carotenogenic genes rather than to the lack of such genes. In addition it has been deduced that different levels of expression

LA0114 LA0134C LA2675 LA1511 LA1937 LA2744 LA1984 LA2879 LA2759 LA2695

96L4891 04L0556 94L1047 90L3840 97L7555 95L3312 96L5367 97L7626 02L7197 04L1844

m. op op. m. op m. op m. sib m. sib m. sib m. sib m. sib m. sib

Taxon

Colour at ripeness

S. S. S. S. S. S. S. S. S. S.

Red Red Red Red Green Green Green Green Green Yellow

pimpinellifolium lycopersicum lycopersicum var. cerasiforme lycopersicum var. cerasiforme peruvianum peruvianum peruvianum chilense chilense chmielewskii

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Table 2 Trolox equivalent antioxidant activity (mmol/kg dw) along the development and ripening of the tomato genotypes analyzed. Stage

TEACmethanol

Total TEAC

Red-fruited accessions 0114 (S. pimpinellifolium) IG 11.11 ± 0.69 MG 10.06 ± 0.56 BR 5.89 ± 0.54 TU 7.99 ± 0.12 RI 46.11 ± 1.52

TEACethyl

18.42 ± 0.22 26.31 ± 1.14 21.50 ± 1.77 21.72 ± 0.89 20.36 ± 1.09

29.53 36.38 27.39 29.71 66.47

0134C (S. lycopersicum) IG 5.94 ± 0.30 MG 8.33 ± 0.86 BR 6.73 ± 0.32 TU 10.04 ± 0.15 RI 51.98 ± 3.44

52.84 ± 5.47 41.01 ± 2.34 35.68 ± 2.92 29.99 ± 1.67 23.19 ± 1.06

58.78 49.33 42.41 40.03 75.18

1511 (S. lycopersicum var. cerasiforme) IG 4.95 ± 0.64 MG 7.31 ± 0.52 BR 15.61 ± 0.49 TU 32.12 ± 2.94 RI 99.24 ± 0.88

48.77 ± 3.45 38.29 ± 3.19 26.47 ± 3.04 26.32 ± 1.01 27.54 ± 2.74

53.72 45.60 42.08 58.43 126.78

2675 (S. lycopersicum var. cerasiforme) IG 7.64 ± 0.00 MG 13.80 ± 0.99 BR 4.96 ± 0.15 TU 6.23 ± 0.18 RI 20.79 ± 1.20

17.76 ± 0.86 19.31 ± 0.46 14.20 ± 0.38 18.42 ± 1.29 19.75 ± 1.98

25.40 33.11 19.15 24.64 40.55

14.68 ± 3.11 22.65 ± 1.43 20.94 ± 3.14

29.80 39.63 43.24

64.96 ± 7.34 59.08 ± 8.86 67.99 ± 5.09

83.65 78.04 85.36

2744 (S. peruvianum) ST1 11.28 ± 1.20 ST2 18.24 ± 1.25 ST3 20.03 ± 0.99 ST4 20.27 ± 3.04

6.89 ± 0.18 22.16 ± 3.33 10.16 ± 0.12 25.38 ± 1.49

18.17 40.41 30.19 45.65

2759 (S. chilense) ST1 15.92 ± 1.07 ST2 16.07 ± 1.66 ST3 12.46 ± 0.82

42.31 ± 1.09 33.67 ± 1.34 82.20 ± 1.76

58.23 49.74 94.66

2879 (S. chilense) ST1 12.74 ± 0.80 ST2 13.48 ± 0.48 ST3 14.65 ± 0.54

50.95 ± 2.76 60.80 ± 9.41 82.96 ± 18.01

63.69 74.28 97.61

Yellow-fruited accessions 2695 (S. chmielewskii) ST1 18.87 ± 1.46 ST2 20.52 ± 1.88 ST3 10.70 ± 0.23

23.47 ± 0.82 26.20 ± 2.20 34.03 ± 1.20

42.34 46.71 44.74

acetate

Green-fruited accessions 1937 (S. peruvianum) ST1 15.12 ± 2.32 ST2 16.98 ± 0.71 ST3 22.29 ± 0.00 1984 (S. peruvianum) ST1 18.69 ± 2.14 ST2 18.96 ± 0.80 ST3 17.37 ± 0.70

25 °C. A mobile phase of methanol (A) and water/methanol (20:80 v/v), containing 0.2% ammonium acetate (B) and methyl tert-butyl ether (C) was used. The gradient elution was as follows: 0–6 min: 95% A. 5% B, 7 min: 80% A. 5% B. 15% C, 12 min: 80% A. 5% B. 15% C, 32 min: 30% A. 5% B. 65% C, 54 min: 30% A. 5% B. 65% C, 56 min: 95% A. 5% B, 62 min: 95% A. 5% B. The flow rate was 1 ml/min. The eluate was monitored continuously from 220 to 780 nm (Dk = 1.2 nm). Identification was achieved by co-chromatography and comparison of the UV–Vis spectroscopic features of the peaks detected with those of reference compounds. The chromatographic and spectroscopic features of the major compounds detected are summarized in Table S1. The reference carotenoids lycopene (w,w-car-

otene) and b-carotene (b,b-carotene) were obtained from HoffmanLa Roche (Basel, Switzerland). Lutein (b,e-carotene-3,30 -diol), violaxanthin (5,6:50 ,60 -diepoxy-5,6,50 ,60 -tetrahydro-b,b-carotene-3,30 diol) and (90 Z)-neoxanthin ((90 Z)-50 ,60 -epoxy-6,7-didehydro-5, 6,50 ,60 -tetrahydro-b,b-carotene-3,5,30 -triol) were isolated from tomato leaves and phytoene (7,8,11,12,70 ,80 ,110 ,120 -octahydro-w,wcarotene) and phytofluene (7,8,11,12,70 ,80 -hexahydro-w,w-carotene) from the Phycomyces blakesleeanus S442 mutant according to standard procedures (Britton et al., 1995). The a-tocopherol, c-tocopherol, chlorophyll a and chlorophyll b standards were procured from Sigma Aldrich (Cambridge, United Kingdom), while pheophytin a and b were obtained by treating acetone solutions of the corresponding chlorophylls with a few drops of 0.1 M methanolic HCl (Khachik et al., 1986; Mendes-Pinto et al., 2005). The levels of isoprenoids were calculated from the corresponding dose–response curves obtained with the corresponding standards. 5.3. Extraction and HPLC analysis of phenylpropanoids and flavonoids Phenylpropanoids and flavonoids were extracted in triplicate from freeze-dried tomato powder (50 mg) into methanol (1 ml) containing 20 lg of salicylic acid as internal standard. The process was carried out at 90 °C for 60 min. Following heating, the suspension was cooled on ice. In order to clarify the mixture centrifugation at 4000 rpm for 10 min was carried out to remove debris. The supernatant was removed and prior to HPLC analysis the mixture was passed through 0.2 ll filters. Aliquots (20 ll) of the extract were injected into the column. The HPLC system used was the same as for isoprenoids. Separations were carried out on a C18 reverse-phase column (250  4.6 mm with 25  4.6-mm guard column from Hichrom, Berks, UK) maintained at 25 °C. The mobile phases consisted of, 2% of methanol in water (v/v) containing 0.015% v/v HCl (A) and acetonitrile (B). The initial composition of the mobile phase was 95% A and 5% B for 10 min. A linear gradient was then applied to 50% B over 50 min. The chromatograms were visualized at 200, 250, 280 and 320 nm (Dk = 1 nm) and the flow rate maintained at 1 ml/min. The identification of chlorogenic acid, chalconaringenin and rutin was achieved by co-chromatography with standards purchased from ExtraSynthese (Lyon, France). Quantitation was carried out from dose–response curves obtained with the corresponding standards. 5.4. Colour measurements performed on the fruit The external colour of the tomato fruit was determined within 2 h of harvest. The measurements were taken at three different points on the equatorial regions of the fruits. The reflectance spectra were obtained using a portable HunterLab MiniScan XE Plus spectrophotometer. The 400–700 nm range of the visible spectrum was recorded with a spectral resolution of 10 nm and a wavelength accuracy of 1 nm. The readings were taken considering a 45°/0° geometry, the CIE Standard Illuminant D65 and the CIE 1964 Standard Observer (10° Observer). For the calibration of the instrument a white and a black reference tiles provided by the manufacturer were used. The colour parameters corresponding to the uniform colour space CIELAB (CIE, 1978) were computed directly by the apparatus. Within the CIELAB colour space a psychometric index of lightness, L*, and two colour coordinates, a* and b* are defined. L* is the approximate measure of lightness which enables each colour to be considered as equivalent to a member of the grey scale, such that it takes values ranging from 0 (black) to 100 (white). The colour coordinate a* takes positive values for reddish colours and negative values for greenish ones, while b* is positive for yellowish colours and negative for the bluish ones. Other psychometric col-

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our parameters, specifically chroma and hue, are defined within the space from the former coordinates. The hue angle (hab) is the qualitative attribute that allows colours to be denoted as bluish, yellowish, reddish, etc., whilst chroma (C ab ) is the quantitative attribute that enables to assess the difference of any given hue relative to a grey colour with the same lightness. The colour differences between tomatoes were worked out as the Euclidean distance between the points corresponding to their colour in the three-dimensional space defined by L*, a* and b*:

DEab ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  ðDL Þ2 þ ðDa Þ2 þ ðDb Þ2

5.5. Determination in vitro antioxidant activity for polar and nonpolar extracts The determination of in vitro antioxidant activity (AA) for both polar and non-polar extracts prepared from the fruit tissue of the wild relatives was carried out according to an ABTS radical cation decolourization assay (Re et al., 1999). This assay is based on the decolourization of 2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) as a result of its reduction in the presence of hydrogen-donating antioxidants in the samples, which can be monitored through the absorbance drop at 734 nm after 6 min of incubation at 30 °C. Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid) was used as the reference antioxidant, enabling the expression of the activities in terms of Trolox Equivalent Antioxidant Capacity (TEAC). The determinations were carried out using aliquots of the same extracts used to determine carotenoids and tocopherols by HPLC-PDA and methanol extracts obtained as for the analysis of phenolics but without the addition of salicylic acid as internal standard. An estimation of the total antioxidant activity (TAA) was expressed as the sum of the AA of the ethyl acetate and methanol extracts. 5.6. Determination of selected gene transcripts encoding carotenoid biosynthetic enzymes The methodology followed that of Fraser et al. (2007). Cloning vectors (pCRII-TOPO, Invitrogen) containing the cDNAs of interest were used for the calibration curves. For this purpose, the transformed plasmids were electroporated into Escherichia coli (DH5a strain), the bacteria grown in agar plates and then in LB media (both containing ampicillin at 50 lg/ml) and finally isolated by the Promega DNA purification system protocol. Typically, RNA was extracted from fruit tissue material frozen in liquid nitrogen upon collection and kept at 80 °C. RNA was extracted by means of the Qiagen RNeasy kit according to the protocol recommended by the manufacturer and including on-column DNase digestion. The extractions were carried out with 100–200 mg of powder material, which was obtained by grinding in a mortar and pestle under liquid nitrogen. Relative gene expression levels were determined using quantitative real-time RT-PCR. The QuantiTect SYBR Green kit (Qiagen) was used routinely and analysis performed on a Rotor-Gene 3000 thermocycler (Corbett Research). RNA (25 ng) in a reaction volume of 20 ll was used. The following oligonucleotides, designed using the Primer3 software (http://primer3.sourcetorge.net/), were added to a final concentration of 0.3 mM: 50 -AGGTATTGTGTTGGACTCTGGTGAT-30 (forward) and 50 -ACGGAGAATGGCATGTGGAA30 (reverse) for actin 50 -TGGCCCAAACGCATCATATA-30 (forward) and 50 -CACCATCGAGCATGTCAAATG-30 (reverse) for Psy-1; 50 GTTGATGGCCCTAATGCATCA-30 (forward) and 50 -TCAAGCATATCAAATGGCCG-30 (reverse) for Psy-2; 50 -TGTTATTGAGGAAGAGAAATGTGTGAT-30 (forward) and 50 -TCCCACCAATAGCCATAACATTTT-30 (reverse) for Cyc-B.

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The following thermocycling conditions were used for the amplifications: 30 min at 50 °C (reverse transcription), followed by 15 min at 95 °C and then 30–40 cycles of 15 s at 94 °C, 30 s at 56 °C and 30 s at 76 °C. Calibration curves were run in parallel with experimental samples, and cycle threshold (Ct) calculations were performed by the Rotor-Gene software (Corbett Research). Normalization of the samples was achieved using actin as a reference. The specificity of the amplifications was verified from the melt curve analysis. Acknowledgements This research was supported by a Marie Curie Intra-European Fellowship (FP6-024671-QUALITOM) granted to A.J.M.M. within the 6th European Community Framework Programme. The authors thank Mr. Chris Gerrish for quality technical assistance and the C.M. Rick Tomato Genetics Resource Center (University of California at Davis) for kindly providing germplasm. Drs. Kaisa Koistenen, and Enrique Sentandreu and Prof. John Halket and are also thanked for their input on the analysis of phenolics. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2010.03.021. References Abushita, A.A., Hebshi, E.A., Daood, H.G., Biacs, P.A., 1997. Determination of antioxidant vitamins in tomatoes. Food Chemistry 60, 207–212. Abushita, A.A., Daood, H.G., Biacs, P.A., 2000. Changes in carotenoids and antioxidant vitamins in tomato as a function of varietal and technological factors. Journal of Agricultural and Food Chemistry 48, 2075–2081. Aksamit-Stachurska, A., Korobczak-Sosna, A., Kulma, A., Szopa, J., 2008. Glycosyltransferase efficiently controls phenylpropanoid pathway. BMC Biotechnology 8. Alba, R., Cordonnier-Pratt, M.M., Pratt, L.H., 2000. Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato. Plant Physiology 123, 363–370. Alba, R., Payton, P., Fei, Z., McQuinn, R., Debbie, P., Martin, G.B., Tanksley, S.D., Giovannoni, J.J., 2005. Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development. The Plant Cell 17, 2954–2965. Alexander, L., Grierson, D., 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. Journal of Experimental Botany 53, 2039– 2055. Azzi, A., 2007. Molecular mechanism of [alpha]-tocopherol action. Free Radical Biology and Medicine 43, 16–21. Baldermann, S., Naim, M., Fleischmann, P., 2005. Enzymatic carotenoid degradation and aroma formation in nectarines (Prunus persica). Food Research International 38, 833–836. Britton, G., Liaaen-Jensen, S., Pfander, H., 1995. Carotenoids. Volume 1A: Isolation and Analysis. Birkhäuser, Basel, Switzerland. Burns, J., Fraser, P.D., Bramley, P.M., 2003. Identification and quantification of carotenoids, tocopherols and chlorophylls in commonly consumed fruits and vegetables. Phytochemistry 62, 939–947. Caris-Veyrat, C., Amiot, M.J., Tyssandier, V., Grasselly, D., Buret, M., Mikolajczak, M., Guilland, J.C., Bouteloup-Demange, C., Borel, P., 2004. Influence of organic versus conventional agricultural practice on the antioxidant microconstituent content of tomatoes and derived purees; consequences on antioxidant plasma status in humans. Journal of Agricultural and Food Chemistry 52, 6503–6509. Carrapeiro, M.M., Donato, J., Goncalves, R.C., Saron, M.L.G., Godoy, H.T., Castro, I.A., 2007. Effect of lycopene on biomarkers of oxidative stress in rats supplemented with [omega]-3 polyunsaturated fatty acids. Food Research International 40, 939–946. CIE, 1978. Recommendations on Uniform Color Spaces, Color-Difference Equations, Psychometric Color Terms, CIE Publication No. 15 (E-1.3.1) 1971, Supplement 2. Bureau Central de la CIE, Vienna. Cremades, O., Parrado, J., Alvarez-Ossorio, M.-C., Jover, M., Collantes de Terán, L., Gutierrez, J.F., Bautista, J., 2003. Isolation and characterization of carotenoproteins from crayfish (Procambarus clarkii). Food Chemistry 82, 559– 566. Davuluri, G.R., van Tuinen, A., Fraser, P.D., Manfredonia, A., Newman, R., Burgess, D., Brummell, D.A., King, S.R., Palys, J., Uhlig, J., Bramley, P.M., Pennings, H.M.J., Bowler, C., 2005. Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nature Biotech. 23, 890–895.

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