## 2 Results
### Expression Profile of Genes of Ascorbate Biosynthesis Enzymes under Abiotic Stresses  
In the current study, three RNA-seq bioprojects regarding pepper leaves were accessed to examine the gene expression pattern under heat, cold, salinity, osmotic, and waterlogging abiotic stresses. In general, all gene members encoding enzymes of D-mannose/L-galactose pathway were ubiquitously expressed, and most of them exhibited high transcript levels, while genes from the alternative pathways generally exhibited lowly expression levels (Figures 6-8 and Tables S6-S8). In heat stress, plants challenged with 40 \({}^{\circ}\)C (PRJNA525913) [38] appeared to strongly affect the Asc biosynthesis by promoting the down-regulation of several genes, including _GMP_, _GME_, _GGP_, _GPP1_, _GalDH_, and _GalLDH_ members from L-galactose pathway during the treatment, while _MIOX3_ and _GalUR_ transcript levels from alternative pathways slightly augmented until 6 h compared to the control (Figure 6 and Table S6). Moreover, the mRNA levels of _PMI1_, _GGP2_, _GPP2_ (12 h), _PMI1_ and 3, and _GallDH_ (24 to 72 h) slightly increased their expression at later periods. Otherwise, the Asc biosynthesis genes in leaves under cold (10 \({}^{\circ}\)C) seemed to be more stimulated until 6 h. Noticeably, _GGP2_ transcripts strongly increased (except in 72 h), followed by the up-regulation of _GME_, _GGP1_, and _GPP1_, with apparent contribution of _MIOX3_ (myo-inositol pathway). Afterwards, most of these genes including _GMP1_ and _GalDH_ experienced a reduction in their expression levels. However, transcript amounts from _PMI_ family and _GallDH_ showed an increase at later exposure times. For leaves treated with NaCl 400 mM, the expression profile of genes associated with Asc biosynthesis suggested the slowdown of L-galactose pathway at all exposure times. Specifically, _GME_ members and _GGP1_ (except in 6 and 24 h) and _GalDH_ members were sharply down-regulated at all times, followed by _GMP1_, _GGP2_, and _GalLDH_ (except at 72 h), mainly at initial and later time points (Figure 6 and Table S6). Relative to alternative pathways, _MIOX3_ slightly decreased their mRNA amounts at 3 h, while _MIOX1_ (24 and 72 h) and _GuILO_ members (72 h) increased. Regarding osmotic stress, the application of mannitol 400 mM did not affect the expression of most genes until 6 h. From 12 h onwards, the expression of _GME_, _GGP_, _GalDH_, and _GalLDH_ families notably decreased, indicating an apparent reduction in Asc biosynthesis. Figure 7 (PRJNA646356) [39] shows the gene expression of Asc biosynthesis pathways evaluated in leaves of two cultivars: a cold-tolerance (A188) and a cold-sensitive inbred line (A122) subjected to low temperature (4 \({}^{\circ}\)C) for 1, 2, and 12 h, and rewarming for 1 h after 72 h cold stress. Overall, tolerant plants (A188) exhibited improved responses by activation of Asc biosynthesis L-galactose pathway at the beginning and the recovery of stress. Several genes, such as _PMI2_ and \(3\), _GMP1_, _GME1_, _GGP1_ and 2, and _GalLDH_ were up-regulated in the tolerant compared to the sensitive cultivar in these exposure times (Figure 7 and Table S7). In this line, transcript amounts of _PMI2_ and \(3\), _GMP1_, _GME1_, and also a member from myo-inositol pathway (_MIOX3_) were notably more expressed in the  
Figure 6: Heatmap illustration of genes encoding ascorbate biosynthesis pathways in leaves of pepper (_Capsicum annuum_ L) under heat (40 \({}^{\circ}\)C), cold (10 \({}^{\circ}\)C), salinity (NaCl\(-\)400 mM), and osmotic stress (manmitol\(-\)400 mM) (PRJNA525913). Up – and down –regulated genes are indicated in red and green colors, respectively, according to Z –score values obtained from three biological replicates. Statistical significance is shown in Table S6. tolerant cultivar at rewarming point. In addition, _PMM_, _GMP1_, _GGP1_ and \(2\), _GME2_, and _GallDH_ genes decreased their expression in recovery, primarily in the sensitive plants.