## 2 Results
### Relative Gene Expression of AsA-related Candidate Genes in Pepper  
Considering the significant differences in AsA concentration, we monitored changes in the expression level of genes involved in the AsA biosynthesis in leaves and during fruit ripening. The comparative analysis was performed considering the PEP10 variety as control genotype, i.e., the gene expression in the PEP1 is relative to that of PEP10, arbitrarily set as 1. Briefly, we analyzed 8 genes involved in the AsA biosynthetic pathways (GDP-mannose pyrophosphylase1 (GMP1), GDP-mannose pyrophosphylase2 (GMP2), GDP-mannose-3'-5'-epimerase (GME), GDP-l-galactose transferase (GGP/VTC2), L-galactose-1-phosphate phosphatase (GPP/VTC4), L-galactono-1,4-lactone dehydrogenase (GalLDH), myo-inositol oxidase (MIOX), and aldo-keto reductase (AKR)) and 6 genes involved in degradation and recycling of AsA (ascorbate oxidase (AO), ascorbate peroxidase1 (APX1), ascorbate peroxidase2 (APX2), ascorbate peroxidase3 (APX3), monodehydroacorbate reductase1 (MDHAR1) and monodehydroacorbate reductase2 (MDHAR2)). We used the beta-tubulin housekeeping gene as an endogenous reference for the normalization of the expression levels of the target genes. The gene expression analysis in leaves indicated the presence of significant differences between the two varieties, with four over-expressed genes in the PEP1 (namely, GMP1, GPP, GalLDH, and AKR) that are all involved in the biosynthetic pathways that lead to the production of L-Galactono-1,4-lactone (Figure 4). Figure 3: Total polyphenol concentration (mg/g FW) during fruit development. Data are mean \(\pm\) s.d. of three biological replicates. IG: immature green; MG: mature green; R: red. At each stage, asterisks indicate a statistically significant difference between varieties (\(\ast\ast\)_p_ \(<\) 0.01; \(t\)-test). On the other hand, the MIOX gene was the most highly under-expressed, suggesting that, at least in this variety, the L-Ascorbic acid biosynthesis from the alternative myo-inositol pathway contributes little to the higher accumulation of ascorbic acid. Among the genes involved in ascorbic acid recycling, the two MDHARs and two APXs coding for enzymes putatively located in the cellular organelles were downregulated in PEP1. On the contrary, the APX2 gene, whose product is predicted to be located in the cytosol, was not differentially expressed. Overall, the gene expression analysis indicated that in the highly accumulating AsA variety (PEP1), ascorbate recycling is reduced. For the analysis of the gene expression during fruit maturation, we compared the two varieties at three stages, IG, MG, and R (Figure 5). The expression pattern in leaves and in fruits was different, further confirming that the ascorbic acid biosynthesis is differentially regulated in different plant organs [30]. Taking into consideration the different stages, seven genes were statistically overexpressed in the PEP1 fruit at immature green, with five genes involved in AsA accumulation (GME, GGP/VTC2, GPP, MIOX, and AKR) and two involved in AsA recycling/oxidation (AO, and MDHAR1). Moreover, IG was the only stage in which GMP1 and GMP2 were not upregulated. Consistent with the observed accumulation dynamics of tAsA, 12 (respectively, 11) genes were upregulated in PEP1 compared to PEP10 at MG (respectively, R). Although the comparison among different maturation stages may be affected by variation in the expression level of the reference gene, it is also evident that the level of overexpression at MG and R was higher compared to IG. In absolute terms, the MG was characterized by the strongest overexpression of several genes (all but AO). This is likely to be needed to support the increasing difference in AsA accumulation between PEP1 and PEP 10 that was recorded during the MG to R transition. During fruit maturation, the expression level of genes involved in the additional pathways for AsA biosynthesis (i.e. MIOX in the myo-inositol pathway, and AKR, in the galacturonic acid pathway) followed the same pattern.