## 3 Discussion
Overall, based on expression level and up-regulation intensity before Asc accumulation, _GGP2_, _GME2_, _GMP1_, _GPP1_, and _GallDH_ may be highlighted as major points and members for the regulation of Asc levels in pepper fruits. In several species, the L-galactose pathway was the primary pathway, and high transcript levels from these families also preceded or coincided with Asc accumulation in fruits of acerola [25; 27], juibe [22], _GPP_ in tomato [44], and _GGP3_ and _GME_ in kiwifruit [13; 49], supporting our findings. Here, stresses simulations affected the gene expression pattern of the principal and secondary pathways in a type- and temporal-specific manner. Climate changes represent a challenge to plant growth and survival, especially under extreme weather events. Ascorbate improves plant resilience to various environmental stimuli by maintaining normal ROS levels [50; 51]. The optimal temperature for pepper normal development ranges from 21 to 28 \({}^{\circ}\)C [39]. In this study, heat and cold treatments provoked an opposite effect in the principal Asc biosynthesising pathway. While high temperature down-regulated the expression of several genes, low temperature increased their mRNA levels, mainly at the beginning and rewarming times (Figures 6 and 7 and Tables S6 and S7). These findings indicate that high temperature severely reduces, but cold increases ascorbate content of pepper leaves, as previously reported in tomato fruit [44], tea [52], and kiwi leaves [53]. Data also suggest that in cold-tolerant cultivar (4 \({}^{\circ}\)C), L-galactose and mynositol pathways activation in the beginning and recovery points indicate that rapid and efficient Asc production may be in the first line for cold response (Figure 7 and Table S7). In this same cultivar, several genes encoding photosystem I and II, cytochrome b6/f complex, and F-ATPase were significantly up-regulated in the tolerant plants at 1 and 2 h of stress simulation [39], pointing that Asc biosynthesis augment was supplied by high photosynthesis flux to overcome cold negative stimuli. For corroboration, the up-regulation of _GPP_[44], _GME_, and _GallDH_ members [54; 55] from the principal pathway in tomato fruits and _GME_ in kiwifruit [53] were also linked to Asc and tolerance gains under cold conditions. The slight up-regulation of specific _MIX_ and _GULO_ members mainly at the beginning (Figures 6 and 7 and Tables S6 and S7) indicate that both stresses may induce alternative pathways in the attempt to maintain or increase Asc pools, though apparently insufficient under heat. Regarding saline and mannitol treatments (400 mM), mRNA amount of the L-galactose pathway reduced along the treatment, while those of the L-glucose and myo-inositol pathways slightly increased at final exposure times (Figure 6 and Table S6). These data show that the induction of alternative pathways is possibly an attempt to balance the slowdown suffered by the principal pathway. In strawberry fruit, drought and salt stresses strongly decreased the ascorbate contents, which were correlated with the down-regulation of _GallDH_[56]. Moreover, low and moderate salt exposures reduced ascorbate levels and _GallDH_ expression levels in leaves and mainly in pepper fruits, from 7 to 21 days of stress, as a result of Na\({}^{+}\) accumulation [57]. For corroboration, high concentration of mannitol increased the oxidized and decreased the reduced form of Asc, while at lower (100 mM)concentration, the Asc content was augmented [58]. Moreover, waterlogging treatment indicated a reduction in the Asc biosynthesis, since various genes from L-galactose pathway were down-regulated in tolerant and sensitive plants, but stimulated at the recovery point mostly in the waterlogging-tolerant cultivar (Figure 8 and Table S8). Waterlogging promotes a hypoxic environment that triggers ROS overproduction [59], verified in the sensitive cultivar.