## 2 Results
### Transcriptional Analysis of Genes Involved in the AsA Metabolic Pathways  
To understand the transcriptional regulation of the main pathways involved in AsA metabolism during development of petals and leaves, and in the flavedo and pulp of fruit of Valencia oranges, the expression of 23 genes corresponding to AsA key metabolic steps were quantified by qRT-PCR [19]. Genes selected included 13 members from AsA biosynthetic pathways; GDP-mannose pyrophosphborylase (_GMP_), GDP-mannose-3-5-epimerase (_GME_), GDP-L-Galactose transferase (_GGP_), L-Galactose-1-phosphate phosphatase (_GPP_), L-Galactose dehydrogenase (_GalDH_), L-galactactone-1,4-lactone dehydrogenase (_GalLDH_), _myo_-inositol oxygenase (_MIOX_), and six isoforms of galacturonic acid reductase (_GalUR_); five genes involved in degradation steps: ascorbate oxidase (_AO_) and four isoforms of ascorbate peroxidase (_APX_); and five genes involved in the recycling steps: two isoforms of dehydroascorbate reductase (_DHAR_) and three isoforms of monodehydroascorbate reductase (_MDHAR_) (Figure 1). Figure 3: Evolution of fruit weight (green) and diameter (red) (**A**), peel color (**B**) and ascorbic acid (AsA) and dehydrofacorbic acid (DHA) contents in the flavedo (**C**) and pulp (**D**) of Valencia Late orange fruit (_Citrus sinensis_) during development and maturation. Fruits were harvested from early development in June (Jn) to over mature fruit in June of the next year (indicated as Jn*). Capital letters indicate significant differences in AsA contents, while lowercase letters indicate significant differences in DHA contents among months for each fruit tissue (\(p\leq 0.05\), Tukey test). Fruit weight (g), diameter (cm), and flavedo color (\(a/b\) Hunter ratio) are the mean \(\pm\) S.E of 20 fruits, whereas AsA and DHA contents (mg/100 g of FW) are the mean \(\pm\) S.E of three replicates. 3.1 Transcriptional Analysis of Genes Involved in AsA Metabolism during Development of Petals and Leaves of Valencia Late Oranges  
Changes in the expression of the selected genes were analyzed in petals and leaves at two developmental stages (Figures 4 and 5). In petals, the expression of most genes increased at anthesis in comparison with those of close flowers (Figure 4). A marked increase (more than 2-fold) was detected in the biosynthetic genes _GMP_, _GPP_, and _GalDH_ of the L-galactose pathway, _GalUR8_, _GalUR10_, _GalUR14_, and _GalUR17_ of the D-galacturonic acid pathway and _MIX_ of the _myo_-inositol pathway (Figure 4A). The gene _GalLDH_ only slightly increased, and a slight decrease was detected for the genes _GME_, _GGP_, and _GalUR5_. With the exception of _AO_ which remained at similar levels of expression, the other four genes of the degradation pathway increased at anthesis. Similarly, three genes of the recycling pathway (_MDHAR2_, _MDHAR3_, and _DHARI_) increased, whereas _MDHAR1_ and _DHARI1_ did not change during petal development (Figure 4B). These results indicate that the stimulation of AsA biosynthesis during petal development was synchronized with the stimulation of degradation and recycling mechanisms as well. Figure 4: Relative expression of genes involved in the biosynthesis (**A**), degradation (**B**), and recycling (**C**) of AsA in petals of closed flowers (CF) and flowers at anthesis (AF) of Valencia Late orange (_Citrus sinensis_). The genes analyzed were _GMP_, _GME_, _GGP_, _GPP_, _GalDH_, and _GalLDH_ of the L-galactose biosynthesis pathway; _GalUR5_, _GalUR8_, _GalUR10_, _GalUR12_, _GalUR14_, and _GalUR17_ of the D-galacturonic acid pathway; _GalUR18_, _GalUR19_, _GalUR17_, and _GalUR18_. biosynthesis pathway; _MIX_, of the _myo_-inositol pathway; _AO_, _APX1_, _APX2_, _APX3_, and _APX4_ involved in AsA degradation; and _MDHAR1_, _MDHAR2_, _MDHAR3_, _DHAR1_, and _DHAR2_, involved in AsA recycling. An expression value of 1 was arbitrarily assigned to the values obtained in the flavedo of fruit in June. Results are the mean \(\pm\) S.E of at least three replicates.