## 3 Discussion  
_Capsicum annuum_ L. is widely recognized as a primary source of vitamin C and spice compounds required for human nutrition [35]. In peppers, ascorbate (Asc) is higher in fruit and accumulates as the ripening proceeds until over-ripe stages [33; 36], similar to tomato and strawberry fruits [23; 44]. The ascorbate accumulation during the maturation of tomato and pepper indicates that the protective function of the antioxidative system plays a fundamental role in the ripening process, given that fruits experience high respiratory rate and ROS production [44; 45; 46]. Nevertheless, the molecular bases responsible for Asc pools regulation in pepper is still unknown despite its global importance and growing number of genomes and transcriptomes sequences available in public databases nowadays. To gain knowledge on molecular mechanisms regarding the significance of Asc biosynthesis pathways and also to detect promising regulatory genes during fruit development and under environmental stresses simulations, nine different transcriptomic datasets were explored. In the present study, we identified 21 genes encoding enzymes of the four proposed ascorbate biosynthesis pathways in pepper. Analyses revealed that these genes are unevenly distributed in 10 out of 12 chromosomes, exposing the structural complexity intrinsic to the biosynthesis of this multifaceted metabolite. Moreover, the transcriptional profile of genes revealed the central contribution of the L-galactose pathway for Asc contents determination in pepper fruit development and ripening, since the expression pattern of genes from alternative pathways did not demonstrate significant association (Figures 3-5 and Tables S3-S5). Previously, the quantification of ascorbate contents for two varieties 'SJ11-3' (higher Asc content) and '06g19-1-1-1' (lower Asc content) showed that it accumulated at breaker, and peaked at ripe stage [36]. Here, we found that all _GMP_, _GME_, _GGP_, _PMI_ and \(2\), and _GPP_2 maintained high expression levels, and were up-regulated at mature green stage (MG) in 'SJ11-3', evidencing their primary role in Asc accumulation. In addition, the up-regulation of _PMI3_, _PMM_, _GGP2_, and _GPP1_ members until breaker stage mainly in SJ11-3 variety seemed to play a key role in Asc pools maintenance (Figure 3 and Table S3). In both of the following experiments, once, all _GMP_, _GGP_, _GME2_, _PMI1_ and \(2\), _PMM_, _GPP2_, and _Gall LDH_ genes were up-regulated at immature or mature green stage in the other five  
Figure 12: Illustration of 50 most abundant cis-regulatory elements identified in theomotor region (1000 bp) of genes encoding enzymes of ascorbate biosynthesis pathways in pepper (_Capsicum annuum_ L.). varieties analyzed (Figures 4 and 5 and Tables S4 and S5). Thus, specific gene member induction at a specific time may reflect the diversity in regulatory mechanisms displayed by each cultivar for fine-tuning of Asc levels. In fact, previous reports also verified higher expression for most of these families at these stages and highlighted the pivotal role of the L-galactose pathway [33; 34], supporting these results. One of the strategies to delay ripening and extend the postharvest time of fruits is the application of nitric oxide (NO), since this molecule inhibits ethylene synthesis and respiration [47]. NO-treated pepper fruit stimulated the L-galactose pathway by up-regulating _GMP1_, _GME2_, _GGP1_ and \(2\), _GPP2_, and _GallDH_ members compared to control (BP1) (Figure 5 and Table S5), indicating that NO treatment improved the antioxidant status by inducing an overproduction of ascorbate. Indeed, NO-treated pepper fruit not only increased the Asc content by 40%, but also glutathione levels and the activities of GaLDH and ascorbate peroxidase [35; 48]. Therefore, NO treatment is a promising alternative for improving the postharvest quality by biofortifying pepper fruit with ascorbate.