Allelic Variation in Paralogs of GDP-l-Galactose Phosphorylase Is a Major Determinant of Vitamin C Concentrations in Apple Fruit

To identify the genetic factors underlying the regulation of fruit vitamin C (l-ascorbic acid [AsA]) concentrations, quantitative trait loci (QTL) studies were carried out in an F1 progeny derived from a cross between the apple (Malus × domestica) cultivars Telamon and Braeburn over three years. QTL were identified for AsA, glutathione, total antioxidant activity in both flesh and skin tissues, and various quality traits, including flesh browning. Four regions on chromosomes 10, 11, 16, and 17 contained stable fruit AsA-QTL clusters. Mapping of AsA metabolic genes identified colocations between orthologs of GDP-l-galactose phosphorylase (GGP), dehydroascorbate reductase (DHAR), and nucleobase-ascorbate transporter within these QTL clusters. Of particular interest are the three paralogs of MdGGP, which all colocated within AsA-QTL clusters. Allelic variants of MdGGP1 and MdGGP3 derived from the cultivar Braeburn parent were also consistently associated with higher fruit total AsA concentrations both within the mapping population (up to 10-fold) and across a range of commercial apple germplasm (up to 6-fold). Striking differences in the expression of the cv Braeburn MdGGP1 allele between fruit from high- and low-AsA genotypes clearly indicate a key role for MdGGP1 in the regulation of fruit AsA concentrations, and this MdGGP allele-specific single-nucleotide polymorphism marker represents an excellent candidate for directed breeding for enhanced fruit AsA concentrations. Interestingly, colocations were also found between MdDHAR3-3 and a stable QTL for browning in the cv Telamon parent, highlighting links between the redox status of the AsA pool and susceptibility to flesh browning.In plants, l-ascorbic acid (AsA; vitamin C) is essential for the detoxification of reactive oxygen species produced under stress or following exposure to pathogens. In addition to these antioxidant functions, AsA has been shown to be involved in a range of important cellular processes, including plant development and hormone signaling, cell cycle, cell expansion, senescence, and as a cofactor for a number of important enzymes (for review, see Davey et al., 2000; Smirnoff et al., 2001; Noctor, 2006). Fruit AsA concentrations have also been correlated with the maintenance of quality during postharvest storage (Davey and Keulemans, 2004; Davey et al., 2007) and have been linked to susceptibility to internal browning in both apple (Malus × domestica; Davey et al., 2006; Davey and Keulemans, 2009) and pear (Pyrus communis; Veltman et al., 1999; Franck et al., 2003). Finally, AsA is clearly an essential dietary component for humans, with a protective role proposed for many disorders and diseases (Diplock et al., 1998). Given its importance for all metabolically active tissues, there is widespread interest in unraveling the mechanisms underlying the genetic control of AsA concentrations in fruits as well as in how AsA interacts with other plant antioxidant pools.The concentration of AsA will be determined by the net rates of biosynthesis, recycling, degradation, and/or intercellular and intracellular transport, but the relative contribution of these various processes depends on several factors, including genetics, tissue type (Bulley et al., 2009), developmental stage (Hancock et al., 2007; Bulley et al., 2009; Ioannidi et al., 2009), and light intensity (Yabuta et al., 2007; Gautier et al., 2009). The biosynthesis of AsA proceeds via l-Gal (Wheeler et al., 1998), although conclusive evidence for all steps has only relatively recently become available (Conklin et al., 2006; Laing et al., 2007). Alternative biosynthetic routes involving uronic acids (Davey et al., 1999; Agius et al., 2003), l-gulose (Wolucka and Van Montagu, 2003), or myoinositol (Lorence et al., 2004) have been proposed in several plant species, including apple (Davey et al., 2004; Razavi et al., 2005; Fig. 1), but their physiological relevance and contribution to the AsA pool is still far from clear in most plant species, with the possible exception of strawberry (Fragaria × ananassa; Agius et al., 2003; Cruz-Rus et al., 2011; Zorrilla-Fontanesi et al., 2011).Open in a separate windowFigure 1.AsA biosynthetic and recycling pathways in plants: l-Gal pathway, reactions 1 to 9; l-gulose pathway, reactions 1 to 5 and 10 to 13; d-galacturonate pathway, reactions 14 to 16; myoinositol/glucuronate pathway, reactions 17 to 21; recycling pathway, reactions 22 to 27. Reactions with question marks are yet to be identified. Numbered reactions are as follows: 1, Glc-6-P isomerase; 2, Man-6-P isomerase (PMI; EC 5.3.1.8); 3, phosphomannomutase (PMM; EC 5.4.2.8); 4, GDP-d-Man pyrophosphorylase (GMP; EC 2.7.7.13); 5, GDP-d-Man 3′,5′-epimerase (GME; EC 5.1.3.18); 6, GDP-l-Gal-phosphorylase (VTC2 or GGP; EC 2.7.7.69); 7, l-Gal-1-P phosphatase (VTC4 or GPP; EC 3.1.3.25); 8, l-Gal dehydrogenase (GalDH; EC 1.1.1.48); 9, l-galactono-1,4-lactone dehydrogenase (GLDH; EC 1.3.2.3); 10, nucleotide pyrophosphatase or sugar-1-phosphate guanyltransferase; 11, sugar phosphatase; 12, sugar dehydrogenase; 13, l-gulono-1,4-lactone oxidase (EC 1.1.3.8); 14, d-galacturonate-1-phosphate uridyltransferase and d-galacturonate-1-phosphate phosphatase (possible); 15, d-galacturonate reductase (GalUR; EC 1.1.1.n9); 16, aldonolactonase; 17, myoinositol oxygenase (MIOX; EC 1.13.99.1); 18, d-glucuronate reductase (EC 1.1.1.19); 19, l-gulonolactonase; 20, d-glucuronate-1-phosphate uridyltransferase; 21, d-glucurono-1-phosphate phosphatase; 22, l-ascorbate peroxidase (APX; EC 1.11.1.11); 23, l-ascorbate oxidase (AO; EC 1.10.3.3); 24, monodehydroascorbate reductase (MDHAR; EC 1.6.5.4); 25, dehydroascorbate reductase (DHAR; EC 1.8.5.1); 26, GSH reductase (GR; EC 1.8.1.7). [See online article for color version of this figure.]As an antioxidant, AsA is able to accept electrons from a wide range of radical substrates, and in this process it becomes oxidized first to monodehydroascorbate and then to dehydroascorbate (DHA). These oxidized forms of AsA can be regenerated by the ascorbate-glutathione (GSH) cycle, so that GSH and the activities of GSH reductase, dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) maintain the size and redox status of the AsA pool (Noctor and Foyer, 1998; Fig. 1). Indeed, overexpression of an Arabidopsis (Arabidopsis thaliana) MDHAR (Eltayeb et al., 2007) and a wheat (Triticum aestivum) DHAR (Chen et al., 2003) have both been shown to increase foliar AsA concentrations in tobacco (Nicotiana tabacum). MDHAR activity has also been positively correlated with both AsA and fruit firmness in tomato (Solanum lycopersicum) after chilling stress (Stevens et al., 2008).Tissue AsA concentrations can also be maintained by intercellular transport, and there is evidence for the long-distance transport of AsA via the phloem from source (leaf) to sink (fruit) tissues (Franceschi and Tarlyn, 2002; Hancock et al., 2003). In apple, fruit AsA concentrations have been suggested to be partly dependent on the translocation of AsA from leaves (Li et al., 2009), but in black currant (Ribes nigrum), others concluded that the contribution of phloem AsA transport to fruit AsA concentrations was negligible (Hancock et al., 2007). While the actual mechanisms of long-distance transport of AsA have not been fully determined, attention has focused on the large family of Nucleobase-Ascorbate Transporters (NATs; de Koning and Diallinas, 2000), and NAT homologs have been found to be highly expressed in vascular tissues (Maurino et al., 2006).Genes involved in several of these mechanisms have been proposed to be key regulators of fruit AsA concentrations, including GDP-l-Gal phosphorylase (GGP) or vitamin c defective2 (VTC2) in kiwifruit (Actinidia deliciosa; Bulley et al., 2009, 2012) as well as GDP-Man-3,5-epimerase (GME; Gilbert et al., 2009), l-Gal-1-P-phosphatase (GPP or VTC4; Ioannidi et al., 2009), and MDHAR (Stevens et al., 2007) in tomato. However, apart from GGP (Bulley et al., 2012), overexpression of these structural genes has to date had limited success in altering the fruit AsA pool (Agius et al., 2003; Bulley et al., 2009; Haroldsen et al., 2011).In this work, we set out to identify potential genetic determinants of fruit AsA concentrations in apple fruit using a combination of molecular and genomic approaches. Initial quantitative trait loci (QTL) analyses of AsA concentrations (Davey et al., 2006) have been expanded to identify QTL for other antioxidants and fruit quality traits over three years, including results in 1 year comparing the concentrations of AsA in fruit and leaves. Alignments of the apple orthologs of genes involved in AsA biosynthesis, turnover, and transport against the whole genome sequence of cv Golden Delicious (Velasco et al., 2010) allowed us to identify candidate genes (CGs) colocating with stable QTL clusters. Using next-generation sequencing (RNA-Seq) data, polymorphic single-nucleotide polymorphism (SNP)-based markers were developed for these colocating CGs, and their positions on individual linkage groups were confirmed by linkage mapping in our mapping population. Finally, associations between allelic variants of these CGs and their expression levels in cultivars with contrasting AsA concentrations allowed us to develop allele-specific markers associated with high fruit AsA concentrations.