## 3 Discussion
Furthermore, it is known that AsA concentrations exert a negative feedback on the expression of _GGP_[52], and this could explain the decrease in transcript accumulation detected in the flavedo of Valencia fruits after reaching the maximum of AsA content in December (Figures 3C and 6A). Beside the relevance of the L-galactose pathway, the elevated expression of _MIX_ in young fruitlets (June)--which drastically decreased towards October--suggest the involvement of this pathway in determining AsA content at the early fruit development stages of Valencia Late oranges (Figures 3C and 6A). In Washington Naved oranges, the involvement of _MIX_ at early stages of fruit development has been also proposed [19]. Thus, it seems that the _myo_-inositol pathway may participate in the synthesis of AsA at the initial stages of fruit development, followed by a major involvement of the L-galactose and D-galacturonic pathway during the last phases of cell enlargement and during the whole period of fruit maturation. In addition to these transcriptional changes, the correlation network of the flavedo of Valencia oranges revealed a negative correlation of AsA content with the _DHAR1_ gene (Figure 8B). Accumulation of the transcript of this gene reached the lowest levels in December (Figure 5C), coincident with the maximum of AsA content (Figure 3C). Thus, it is likely that the expression of _DHAR1_ in coordination with other genes involved in AsA degradation may be part of the mechanisms to maintain the homeostasis of the AsA pool in the flavedo of fruits. Regarding the transcriptional regulation of AsA in the pulp, the correlation network revealed that most of the biosynthetic genes of the D-galacturonic pathway (_GalUR5_, _GalUR10_, _GalUR12_, and _GalUR17_), and also _MIOX_ and _GalDH_, were negatively correlated with AsA concentration. Moreover, _GalUR12_, _GalUR17_, _GalDH_, and _GME_ were also negatively correlated with DHA content (Figure 8D). The fact that most of these genes were downregulated, whereas AsA experienced slight variations suggest that the transcriptional regulation of these pathways is not playing a relevant role in AsA accumulation. Other genes of AsA degradation (_APX1_, _APX3_, and _AO_) were also negatively related to AsA content, contributing also to the maintenance of the homeostasis. Finally, a close examination of the expression of most genes of the L-galactose and the recycling pathways, as well as _APX2_ and _APX4_ of the degradation pathway, revealed a similar transcriptional profiling (Figure 7). Taken together, these results indicate that the regulation of AsA content in the pulp of Valencia orange fruits may be the result of a complex and coordinated balance between biosynthetic, degradation, and recycling pathways, contributing to keeping the content of this antioxidant relatively stable during fruit development and maturation. Analysis of the co-expression of genes of the AsA metabolic pathways and the search of _cis_-elements in the promoter region of these genes can help to decipher common regulators of the process and to understand the mechanisms and environmental signals involved in the synthesis and accumulation of AsA. Up to now, there are only few TFs known to affect either positively or negatively AsA metabolisms in plants, as HDZIP1 in tomato fruits [32], and ERF98 and AMR1 in Arabidopsis [53], but none has been so far described in _Citrus_. The in silico analysis of the promoter regions of the 23 genes of AsA metabolism analyzed revealed interesting features (Figure 9). GT factor binding motifs are present in all the genes analyzed. These motifs have been found in the promoters of a number of plant genes, but their regulatory function of these TFs remains unknown [54]. Moreover, ABA and sugar responsive elements (ABRE, STK, and SURE) where found in the majority of the genes, indicating that the regulation of AsA metabolism may be mediated by this phytohormone and sugars.