Regulation of fruit vitamin C metabolism

L-Ascorbic acid (AsA), or vitamin C, is an abundant low molecular dietary antioxidant compound that exerts a protective role against chronic diseases and disorders in humans. Genetic control of AsA concentrations in fruits can involve biosynthetic, recycling and transport mechanisms, as well as being linked to changes in whole fruit metabolism and/or plant growth. The aim of this work was to understand the genetics underlying the control of vitamin C metabolism in fruits. Two major approaches have been employed using two different fruit crops tomato and apple. Both species have medium AsA levels, but the significant variation among different cultivars (particularly in apple) clearly indicates that there is potential for improvement of this trait. In the first phase of this work, extensive biochemical analyses, as well as 14C-labelled and non-labelled substrate feeding experiments were carried out in various tissues and fruit ripening stages in two tomato cultivars. Results confirm that the L-galactose pathway is the main AsA biosynthetic route in tomato fruits, but substrates from alternative pathways can also be converted to AsA in fruits at specific developmental stages and genotypes. Overall, both biosynthetic capacity and the turnover rate of the AsA pool decline throughout ripening. However, the two tomato cultivars (AsA-rich Santorini and AsA-poor Ailsa Craig ) exhibited different profiles of AsA accumulation during ripening, but a characteristic peak in AsA-totAsA concentrations at the breaker stage. Differences in the activity of AsA recycling enzymes (particularly of MDHAR) helped explain the variations in fruit AsA concentrations of the two cultivars. In addition, expression profiles of an orthologue of GDP-L-galactose phosphorylase (SlGGP1), and two gene copies of monodehydroascorbate reductase (SlMDHAR1 and SlMDHAR3) were all closely associated with changes in AsA concentrations throughout ripening. Among these candidate genes, the expression of SlGGP1 was closely related with high AsA concentrations at red ripe fruits. To investigate the role of ethylene, two mutants inhibited in their ethylene responses (Never-ripe and ripening-inhibitor) were also studied. Results clearly indicate a key role of ethylene receptors in the control of fruit AsA pool that should be further investigated. In parallel with the tomato work, a combination of molecular, genetic and genomic approaches (QTL mapping, NGS, SNP discovery, gene expression and allelic association studies) were used to help identify key genes regulating fruit AsA concentrations in apple tissues. Stable AsA-QTLs over two or three years of measurements were mapped in LGs 10, 11, and 17. Candidate gene mapping demonstrated that the three paralogues of MdGGP are key regulators of AsA and totAsA concentrations in mature apple. Further, allelic variations in the transcript sequences of MdGGP1 (LG 11) and MdGGP3 (LG 10) arecloselylinked to fruit totAsA concentrations both within the Telamon x Braeburn mapping population and across a selection of commercial cultivars. Results from gene expression studies (qPCR) confirm that the expression of MdGGP1 and MdGGP3, as well as of a paralogue of dehydroascorbate reductase (MdDHAR3-3), are all strongly associated with fruit AsA concentrations in a selection of high- and low-vitamin C cultivars. These SNP-based markers are thus potentially excellent candidate for use within breeding programs to screen for progeny with increased AsA concentrations. At last, MdDHAR3 was found to have a key role in regulating the redox state of the AsA pool in apple fruits, and increased flesh DHA concentrations are associated with susceptibility to flesh browning. The separation of fruit into green and red side is beneficial for the better understanding of fruit AsA regulation, as highlighted by the improvement of AsA-QTL size and properties. The combined results in tomato and apple allow us to suggest that GGP has a central role in regulating fruit AsA levels which is broadly applicable at different fruit species. In terms of applied research, the identification of novel genes/alleles linked to high fruit AsA concentrations in apple can have immediate applications within fruit breeding programs via marker assisted breeding and screening.<w:latentstyles <