Transcriptional regulation of the vitamin C metabolism in apple fruit (Malus × domestica)

The saying 'an apple a day keeps the doctor away' accurately summarizes that apples are full of healthy compounds. Vitamin C (ascorbic acid, Asc) is one of those compounds and is particularly interesting because it acts both as a vitamin and as an antioxidant. Since vitamin C is not only crucial for human nutrition, but can also improve the storability of apples and their resistance against (a)biotic stresses, vitamin C content is an interesting trait for apple crop improvement. With the aim of breeding high vitamin C apple cultivars it is important to get an insight in the natural biodiversity of vitamin C content in apple fruits. Therefore, in this PhD, the ascorbic acid (Asc), dehydroascorbic acid (DHA), and total Asc (Asc + DHA) concentration in the apple pulp of 79 old and recent cultivars at harvest was assessed. This revealed significant variation in the Asc (21‑fold), DHA (149-fold) and total Asc (11-fold) content of the fruit flesh, indicating a large underlying genetic biodiversity. A population structure analysis, based on 8K SNP chip high density genotyping, identified two groups in this germplasm, one group with 21 elite accessions and a second group composed of 58 local/noncommercial accessions. The average (total) Asc content in the pulp of local and elite varieties was similar, probably as a consequence of the lack of targeted selection for this nutritional aspect in apple. Out of the 79 apple cultivars screened, ten genotypes with either the highest or the lowest concentration of total Asc in de pulp at harvest were used for monitoring vitamin C dynamics during fruit development and subsequent cold storage and shelf life. Regarding the Asc/DHA ratio, the balance generally tilted towards the reduced form (Asc) during fruit development, while there was an evolution to a more oxidized (DHA) state during storage. For each of the ten assessed cultivars, the apple peel had a higher vitamin C concentration than the apple pulp at any point during fruit development and storage most likely because of its photosynthetic activity and more direct exposure to abiotic and biotic stresses (irradiation, ozone, pathogens,…), events that produce high levels of ROS that need to be fine-tuned by antioxidants such as vitamin C. A correlation analysis showed that the amount of total Asc in the apple pulp positively correlated with that in the peel throughout fruit development and storage of the ten selected cultivars. Gloster, Nicogreen and Marie Joseph d'Othée (MJD) showed the most distinct course of Asc content in the apple fruit flesh during fruit development and subsequent cold storage out of the ten genotypes for which the vitamin C dynamics was monitored. Moreover, from mid-summer onwards the three cultivars consistently had different amounts of Asc, with MJD having the highest content, followed by Nicogreen, in turn followed by Gloster. Therefore, Gloster, Nicogreen and MJD were selected to perform an RNA-sequencing analysis of fruit flesh samples at four time points during fruit development, i.e., after June drop, in the middle of summer, at the beginning of autumn and at harvest, and after subsequent cold storage. This experimental set-up allowed to investigate the transcriptional regulation of the Asc metabolism in multiple cultivars over the apples' complete life cycle. The existence of four different biosynthesis pathways combined with the interplay of biosynthesis, recycling and oxidation routes makes the Asc homeostasis particularly complex. There is tremendous ambiguity in the literature about to what extent the transcriptional regulation of these different pathways contributes to the Asc concentration in apple pulp, and whether this is time- and/or genotype-dependent. Hence, firstly, this PhD focused on the expression of the known Asc metabolic genes to unravel the dynamics and relevance of the different Asc pathways in apple fruit flesh. The results indicated that the D-mannose/L-galactose and D-glucuronate biosynthesis pathway together with the Asc recycling and oxidizing pathways were transcriptionally activated during fruit development and cold storage and hence likely participate in the Asc homeostasis in the apple pulp. None of the involved Asc metabolic gene families were differentially expressed between Gloster, Nicogreen and MJD during fruit development, indicating a conserved transcriptional regulation of the different Asc metabolic enzymes. The correlation between Asc dynamics and the expression of Asc metabolic gene families, on the other hand, was cultivar- and time-specific. Unlike the Asc levels, DHA levels did seem to be controlled by the transcriptional amounts of Asc metabolic genes in all three cultivars. Interestingly, the expression of AO15.1, encoding an Asc oxidizing enzyme, positively correlated with the DHA concentration in all three cultivars and therefore was the only common Asc metabolic gene of which the expression level seemed to be key for the Asc oxidation level. In addition, the RNA-seq data was used to identify putative upstream regulators of the Asc metabolism in apple fruit pulp. Nine putative apple transcription factors were identified that seem to control the transcript abundance of specific Asc metabolic genes in Gloster, Nicogreen and MJD during fruit development and subsequent storage. MYB5.11b, four Dof22 isozymes and HSF24 are apparent positive transcriptional regulators of Asc metabolic genes in apple since the expression dynamics of their genes was strongly positively correlated with the expression profiles of genes encoding the Asc biosynthesis enzymes GGP, GPP, GDH and/or the Asc recycling enzyme GR. In contrast, the putative regulators nsLTP1.11, AS1 and MYC2 probably have a negative effect on the size of the reduced Asc pool as the expression of the corresponding genes correlated strongly positively with the expression of genes from the Asc oxidation gene families, AO and APX. The expression of AS1 correlated positively with the expression of AO15.1 in all cultivars, indicating that not only the influence of the transcriptional abundance of AO15.1 on the Asc metabolism is conserved in the pulp of on-tree and stored apple fruits, but also the upstream transcriptional regulation of AO15.1 itself. The Myb5, Dof22 and nsLTP1 genes are orthologs of genes encoding transcription factors that are known to influence the Asc content in other (model) crops, which reinforces the hypothesis that their gene products affect Asc metabolism in apple as well. Finally, a differential gene expression analysis was performed to find genes in the apple fruit flesh that are differentially expressed over time and between cultivars and therefore could be transcriptionally regulated by the (total) Asc concentration. There were no differentially expressed genes of which the expression correlated in the same way with the (total) Asc concentration in all three assessed cultivars, suggesting that the influence of vitamin C on gene transcription is not conserved in apple pulp. Together with future studies about protein abundances and enzyme efficiencies of the Asc metabolic gene products, this transcriptomic research could unravel the complex regulation of the Asc metabolism in apple fruit pulp. Moreover, by putting forward several candidate Asc metabolic genes and transcription factors, this study sets the stage for further validation of the importance of these candidates and ultimately opens the road to breeding for commercial apple cultivars with an increased Asc level in the fruit pulp.

Jaar van publicatie:2021

Toegankelijkheid:Closed