El Conocimiento Pedagógico del Contenido desde la perspectiva de

Consumers often associate fruit colour with flavour, safety, storage time, nutrition, and level of satisfaction (Pedreschi et al., 2006). However, this judgment of quality by fruit colour may not necessarily be right for many fruit and vegetables and can be misleading indicator of quality (e.g. refrigerated supermarket red tomatoes with lack of flavour) (Shewfelt, 2002). Tomatoes harvested for fresh consumption are often picked at mature- green or early ripe stages and transported to retailers at low temperature (Chomchalow et

15 al., 2002). Depending on temperature uneven blotchy red colouration or complete failure of red colour development in tomatoes can be induced (Cheng and Shewfelt, 1988; Lurie et al., 1996). For instance, at 2 °C, one of the most obvious changes in mature-green tomatoes is complete failure to ripen (Hobson, 1987). Fruit locules remain green and seeds turn brown (Moline, 1976). Storage at 2 or 6 °C can inhibit full red colour development in tomatoes harvested even at advanced maturity stages (i.e. breaker or pink) (Ilic and Fallik, 2005; Gómez et al., 2009).

Tomato colour changes from green to red during normal ripening as chloroplasts transform into chromoplasts, chlorophyll degrades and lycopene, a major carotenoid responsible for red colour development, accumulates (Shewfelt, 2002). During low temperature conditions modification of red colour development may be because chloroplasts are the first organelles that undergo structural changes (Marangoni et al., 1989; Yang et al., 2009). Ultrastructural observations indicated that failure to ripen was due, in part, to interruption in conversion of chloroplasts to chromoplasts while non-chilled fruit showed lycopene crystals in healthy plastids (Moline, 1976). Rugkong et al. (2011) suggested that loss of chlorophyll in tomato during cool storage was manifested as yellowing. Decreased levels

of chlorophyll in chilled tomatoes probably unveil β-carotene, causing the appearance of yellow blush colour (Dodds et al., 1991). Chilling may also have caused accumulation of chalco-naringenin, a yellow compound found in tomato pericarp (Baker et al., 1982 as cited in Dodds et al., 1991).

The physiological abnormalities associated with blotchy red colouration could be due to abnormal functioning of random patches of tissues. It is believed that CI is not translocatable (Saltveit and Morris, 1990). Eaks and Morris (1957) found that CI symptoms in cucumber were localised to the half of an intact fruit which was exposed to chilling. Equally, failure of uniform heating may induce differences in CI and colour development between heated and non-heated halves of a tomato (Lu et al., 2010) or an avocado (Woolf, 1997), indicating a localised rather than systemic effect of heat treatment on postharvest quality parameters of tomatoes (Lu et al., 2010). It is possible that uneven red colouration in tomatoes is due to localised failure of patches of tissues to develop red colour. Alternatively, it may be possible that blotchy red colouration is not random rather it is a patterned disruption of normal ripening prototype. Tomato fruit is composed of distinct tissue types including pericarp, placenta, septa and locular gel tissues (Brecht, 1987).

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Locular gel develops prior to ripening of the pericarp (Kader and Morris, 1976). Ripening initiates in mature-green tomatoes in locular gel, proceeds through placenta to the columella, with the first visible sign of red (yellow or orange) pigmentation at the blossom end and colour then progresses towards stem end of the fruit (Yahia and Brecht, 2012). A climacteric rise in ethylene production has also been observed in gel tissue prior to other tissue types (Brecht, 1987). It is possible that chilling affects tissues differentially resulted in different ripening behaviour of various tissues and eventually uneven blotchy red colour appears. Additionally, Jiang et al. (1999) observed that 1-MCP treated bananas showed uneven skin de-greening and the authors attributed this to positional differences in the rate of new synthesis of ethylene binding sites. This may also be true for tomato.

Molecular studies reveal that the interruption of gene expression involved in colour development results in altered colour development in chilled tomatoes. Lycopene is an intermediate of the carotene biosynthetic pathway in tomato (Figure 1.3). Carotenoid formation utilizes the ubiquitous isoprenoid precursor geranylgeranyl pyrophosphate (GGPP). Two molecules of GGPP are condensed to form phytoene and this reaction is catalysed by phytoene synthase (PSY). Phytoene is then converted to lycopene in a series of dehydrogenation reactions, which introduce four double bonds into the phytoene

molecule. This conversion is performed by the sequential action of phytoene and ζ-

carotene desaturase enzymes respectively. β-carotene and α-carotene are then synthesised by the action of the enzyme sesquiterpene cyclase (Alexander and Grierson, 2002). As fruit ripen, the concentration and activity of sesquiterpene cyclase are reduced; leading to the accumulation of lycopene in the stroma and thus red colour develops in tomato fruit (Paliyath and Murr, 2008).

Lurie et al. (1996) reported that phytoene synthase1 (PSY1) gene which encodes fruit specific phytoene synthase was down-regulated during chilling. Bird et al. (1991) observed that down-regulation of PSY1 resulted in yellow tomato devoid of lycopene. Not only PSY1 gene, but also that gene expression of at least three other enzymes involved in carotenoid biosynthesis (carotenoid isomerase-CRTISO, geranylgeranyl diphosphate synthase 2-GGPPS2 and 1-deoxy-D-xylulose-5-phosphate synthase-DXS) were down- regulated in chilled tomatoes (Rugkong et al., 2011). They further indicated that down- regulation of a MADS-box transcription factor necessary for fruit ripening, LeMADS-RIN (responsible for conferring nonripening phenotype of the rin mutant) was down-regulated

17 after 4 weeks chilling at 3 °C and this reduced expression contributed to the chilling- induced delayed ripening.

GGPP 1 Phytoene 2 ζ-Carotene 3 Lycopene 4 5 α-Carotene β-Carotene 7 6 Lutein Zeaxanthin

Figure 1.3 Summary of the biosynthetic pathway for carotenoids. Numbers

indicate enzymes responsible for the conversion. 1. Phytoene synthase. 2. Phytoene desaturase. 3. ζ-Carotene desaturase. 4. β-Cyclase. 5. β- and ε-Cyclase. 6. β- Hydroxylase. 7. β- and ε-Hydroxylase (Source: Alexander and Grierson, 2002).

While reduction in expression of carotenoid synthesis genes during cool storage results in altered red colour development in chill-induced fruit, down-regulation of gene expression is possibly a function of chilling durations to which fruit are exposed. For example, Rugkong et al. (2011) found that tomatoes stored at 3 °C for 4 weeks showed reduced expression of PSY1 and CRTISO compared to tomatoes at harvest. Expression of these genes increased after fruit were transferred to 20 °C following 1 week at 3 °C. When fruit were stored for 2 weeks, they showed increased expression of these genes during ripening at 20 °C but the expression was lower than in fruit stored for 1 week. Similarly, down- regulation in GGPPS2 expression was observed with a longer chilling period. These results agree with the idea that for a short duration at low temperature tomato may be able to develop red colour whereas for longer duration tomato may fail to do so and instead show uneven blotchiness or yellow colouration.

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A phenotype that shows uneven-blotchy red colouration during normal ripening has also been reported, although the cause of blotchy ripening is not clearly understood (Dr. J. Giovanonni, personal communication, Ethylene 2012). Blotchy areas are usually confined to the outer pericarp walls, but radial walls can also be affected in extreme cases (Yahia and Brecht, 2012). Some pre-harvest factors include low light intensity, cool temperatures, high soil moisture, high nitrogen, and low potassium or combinations of these factors, are thought to contribute to blotchy ripening (Yahia and Brecht, 2012). Blotchy areas of fruit walls contain less organic acids, dry matter, soluble solids, and starch sugar (Adams et al., 1978), indicting some kind of disturbed metabolism. Molecular work suggested that over- expression of a fruit ripening booster (FRB) gene, an auxin response factor, caused accelerated and patchy ripening (Breitel, 2012). Down-regulation of DR12 (developmentally regulated clones), another auxin-response-factor homolog, in the tomato resulted in a pleiotropic phenotype including dark green and blotchy ripening fruit (Jones et al., 2002). While over-expression of some genes may cause a blotchy phenotype, it remains unclear whether blotchy uneven ripening induced by CI is related to expression of those genes. Blotchy ripening caused by FRB appeared to have quite sharp boundaries between green and red tissues (Breitel, 2012), unlike CI where the colour was more diffused with patches ranging from yellow to red on fruit surface.

Overall, when tomato is exposed to low temperature for a certain period of time, fruit exhibits abnormal colour development. Various factors may play a role in inducing blotchy colour development including modification of organelles such as chloroplasts or the inhibition of enzymes activities in biosynthetic pathway to lycopene or degradation of chlorophyll, down-regulation of genes encoding those enzymes or over-expression of some genes related to blotchiness.