ESTRUCTURA Y COMPETENCIAS DE LA JUNTA DE CASTILLA Y LEÓN PARA LAS POLÍTICAS EN IGUALDAD Y

5.1.1 Harvest maturity for genotypes from interspecific crosses

Working with breeding populations of fruit trees consisting of hundreds of different genotypes is generally challenging for achieving the conditions ideal for typical postharvest experiments. The genetic makeup of the pear samples is complex because of their interspecific origin (Figure 2.1). European pear requires low temperature conditioning for initiation of ripening whilst Asian pears generally ripen on the tree (Kingston, 1992). Fruit samples after harvest were stored for up to 3 months with later exposure to room temperature for at least 24 hours. It is likely that fruit samples with more Asian characteristics do not require long term exposure to chilling temperature (0-1°C) and were already over mature when assessed for FD. However, given that it would be impossible to establish harvest maturity indices for each genotype, there seems no better option than what was chosen here. Fruit were harvested on the basis of visible cues related to maturation: i.e. sequential harvests (whenever possible) commencing when fruit drop was first seen for a genotype, and selecting individual fruit showing a change in background colour from deep green to light green/yellow.

Given the complexities posed by interspecific populations for accurate maturity determination, crosses within species are recommended for studies of postharvest traits, especially in pear. Independent multiple harvests should be assessed for 159

complex quantitative traits that are known to be influenced by harvest date (maturity stage) and, as was done here, maturity-related data (e.g. soluble solids content, dry matter and firmness) should be captured to allow post hoc analysis of possible confounding influences from under- or over-maturity.

5.1.2 Do skin properties predispose fruit to FD?

Loss of compartmentation as a result of mechanical damage leads to the enzymatic oxidation of phenolics in damaged pear fruit. Since the skin appearance of different varieties can vary markedly, several authors have investigated whether some attributes of the skin may explain varietal differences in susceptibility (Amarante et al., 2001a; Palmer et al., 2008). Those studies have demonstrated that there can be significant variability in the skin of even a single commercial cultivar (Palmer et al., 2008) which may contribute to fruit-to-fruit variation in FD susceptibility. In current study, a total of 40 fruit (20 low and 20 high FD category) were tested for role of skin properties in FD susceptibility. No consistent differences were observed between the two groups of genotypes. This does not mean that the skin is unimportant to the development of FD; but it is clear that we do not yet have any particular skin attributes that appear to predispose the fruit surface to FD.

5.1.3 Role of phenolics as antioxidants in the incidence of FD

Phenolics are a wide spread group of secondary metabolites in almost every plant species and their quantitative distribution may vary from organ to organ within and between plants of the same species. Researchers have tried to correlate tissue browning to the amount of substrate (phenolics) or enzyme (PPO) activity of the fruit. However contradictory conclusions have been reached by different authors which illustrates the complexity of the browning mechanism. Interestingly, no preliminary literature is available regarding role of phenolics in FD susceptibility in Asian pears, with all published papers referring only to European pears (Meheriuk et al., 1994; Amiot et al., 1995; Bertolini and Trufelli, 2002; Hamauzu and Hanakawa, 2003; Burger et al., 2005). Although an overall weak negative correlation was identified in both populations between phenolic content and FD score, different trends were observed in individual genotypes. Phenolic compounds such as caffeic

esters and catechin act as good antioxidants as well as good substrates for browning processes. At relatively low concentrations they act as pro-oxidants for initiation of browning while at higher concentration they act as antioxidants (Fukumoto and Mazza, 2000).

Phenolics can prevent enzymatic browning by reacting with oxygen, with intermediate products by breaking the chain reaction or by acting as chelating agents and reducing Cu++ to Cu+ (Lindley, 1998; Li et al., 2011; Loannou, 2013). Phenolics are also known to interact directly with polyphenoloxidase (PPO) which can lead to inhibition of PPO activity (Le Bourvellec et al., 2003). Le Bourvellec et al. (2003) studied the inhibitory effect of caffeoylquinic acid, epicatechin and procyanidin oxidation products; they oxidized the mixture of caffeoylquinic acid and epicatechin by reacting with caffeoylquinic acid o-quinone to get oxidized products. Oxidized products from all compounds inhibited PPO activity and regenerated the original phenolic substrates. The related phenomenon of ‘coupled oxidation’ may occur in which the product (quinones) of the first step of oxidative browning further oxidizes other phenolics like flavanols which results in the regeneration of the original phenolics (Fig. 5.1, (Nicolas and Potus, 1994). Chlorogenic acid is reported to play a prominent role in the oxidative degradation of other phenolics with regeneration of chlorogenic acid itself. Reaction products resulting from chlorogenic acid oxidation are light to colourless in hue while quinones form darker compounds (Rouet-Mayer et al., 1990; Goupy et al., 1995).

Chlorogenic acid constituted the major phenolic in the studied genotypes. There was no clear relationship between chlorogenic acid and FD score in 2011 and 2012 while in 2013 a negative correlation of 0.46 (P<0.05) was observed. It can be speculated that in this study, phenolics actively participated as antioxidants during enzymatic browning to reverse the o-quinones back to original phenols as well as acting as substrates for PPO. The antioxidant effect may explain the overall weak negative correlation between phenolic concentration and FD across the wide range of genotypes in this study.

Ascorbic acid (AsA) was included in this study because of its antioxidant role. Quinones from oxidative reactions can react with ascorbic acid and result in 161

regeneration of initial phenols (Figure 5.1). Browning can be stopped until ascorbic acid is consumed or depleted then formation of brown pigments will again take place (Rouet-Mayer et al., 1990). However, in this study there were no correlations between ascorbic acid concentration and FD or any other parameter. During long term storage, AsA can be oxidized to dehydroascorbic acid (DHA) (Mazurek and Pankiewicz, 2012). Only total ascorbic acid was analysed in this study. So there is a possibility that variable amounts of AsA were oxidized to DHA during storage in the various genotypes. This could be verified in future work.

Figure 5.1: Enzymatic browning reaction catalysed by Tyrosinase (PPO) enzyme. Ortho-quinones reduced back to original phenols as a reaction of coupled oxidation or reducing agent. CGA: chlorogenic acid, AsA: Ascorbic acid.

Measurement of antioxidant activity is recommended to estimate the antioxidant effect of phenolics and AsA in context of enzymatic browning. Assays for

T y ros inas e + O 2 OH R Monophenol Colourless OH OH R Diphenol, CGA Colourless O O R O- quinone Browning pigments Tyrosinase+ O2 AsA, Catechin Melanin Coloured Amino acids, proteins Phenolic species Condensation products

antioxidant activity like FRAP (ferric reducing antioxidant power) and ORAC (oxygen radical absorbance capacity) are suggested due to their wide range of applicability (Cao et al., 1993; Benzie and Strain, 1996).

One final consideration relating to metabolite concentrations in this study is to note that metabolites were sampled 24 hours after FD initiation on each pear, using fruit skin from non-damaged parts of each fruit. It is possible that the FD assay could cause a systemic change in phenolic concentrations around undamaged portions of the fruit (Saltveit, 2000). One way to avoid this problem would be to take larger numbers of fruit for analysis (which would require older trees than were used here). So metabolite samples could be taken from undamaged fruit and FD assessments could be made on matching batches of fruit. Nevertheless, using the identical fruit for both FD and metabolite assessment still seems the best place to start.

5.1.4 Robustness of detected QTLs

A large number of QTLs for pear fruit quality traits including FD were identified. FD is a complex disorder and has been known to be influenced by genetic x environment (GxE) interaction as described in chapter 2. Besides these variations a number of QTLs were detected associated with FD of which two were stable across the years (2). Robustness of the QTLs can be tested by repeating the study in different seasons, at different growing environments and across the populations. In this study phenotypic data for POP369 were collected in two years (2011 and 2012) which provided confidence for QTL robustness in this population. QTLs from POP356 were considered real if they were reproducible across the populations. Practical implications of these stable QTLs are presented in section 5.3 in this chapter.