## 2 Results
### Expression Profile of Genes of Ascorbate Biosynthesis Enzymes during Fruit Development  
In the current study, three public RNA-seq bioprojects regarding pepper fruit development and ripening were selected to examine the expression pattern of genes encoding Asc biosynthesis. In general, all gene members encoding to the D-mannose/L-galactose pathway were ubiquitously expressed, and most of them exhibited high expression levels. In contrast, genes of the alternative pathways exhibited low expression levels or were even unexpressed (_GullLO1_ and _MIOX2_ and _4_) in these experiments (Figures 3-5 and Tables S2-S5). Figure 3: Heatmap representation of genes encoding enzymes of ascorbate biosynthesis pathways in fruit pericaps of \({}^{\prime}\)SJ11\(-\)3\({}^{\prime}\) and \({}^{\prime}\)06g19\(-\)1\(-\)1\(-\)1’ pepper (_Capsicum annuum_ L.) varieties at 20, 30, 40 and 50 Days After Anthesis (DAA) (PRJNA485468). 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 S3. In experiment 1 (PRJNA485468) [36], the transcriptome of fruit pericarps of two pepper varieties 'SJ11\(-\)3' (higher Asc content) and '06g19\(-\)1\(-\)1\(-\)1' (lower Asc content) at the immature green (IG; 20 Days After Anthesis--DAA), mature green (MG; 30 DAA), breaker (BR; 40 DAA), and mature red (MR; 50 DAA) stages was analyzed (Figure 3 and Tables S2 and S3). Among the genes analyzed from the D-mannose/L-galactotes pathway in the variety SJ11\(-\)3 (higher Asc content), all _GMP_, _GME_, _GGP_, _PMI1_ and \(2\), and _GPP2_ members exhiited higher expression level or were up-regulated at MG stage, preceding the overproduction of Asc. While the 06g19\(-\)1\(-\)1\(-\)1\(-\)1 variety, mostly revealed lower levels of these transcripts and a continuous decrease from IG until MG stage. _PMI3_, _PMM_, _GGP2_, and _GPP1_ genes were up-regulated until breaker stage for both varieties. However,  
Figure 4: Heatmap representation of genes encoding enzymes of ascorbate biosynthesis pathways in fruit peels of four pepper (_Capsicum annuum_ L.) varieties, HJ10\(-\)1, HJ11\(-\)3\(-\)1, CJ12\(-\)17\(-\)1, and 0622\(-\)1\(-\)3\(-\)2\(-\)1\(-\)3\(-\)1, at 30 and 50 DAA (PRJNA533286). 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 S4. SJ11\(-\)3 exhibited higher transcript amounts and peaked at breaker stage, when Asc increase was maximum in both varieties. In addition, transcript levels of _GMP1_ and _GalDH_ were higher in 06g19\(-\)1\(-\)1\(-\)1 from MG to MR. Overall, the expression of genes from alternative pathways did not seem to correlate with Asc accumulation at later stages. _GullO2_ (L-gulose pathway) expression was significantly higher in SJ11\(-\)3, mainly at IG and MR stages, while _GalUR_ transcripts (D-galacturonate) showed higher amounts in IG and were absent in MR stage of SJ11\(-\)3 compared to 06g19\(-\)1\(-\)1\(-\)1. _MIOX_ gene expression profile was similar in both varieties: _MIOX3_ was more expressed at IG, while _MIOX1_ was only expressed after the MG stage. Figure 5: Heatmap representation of genes encoding enzymes of ascorbate biosynthesis pathways in fruit of California\(-\)type sweet pepper (_Capsicum annuum_ L.) at immature green (IG), breaking point (BP), and ripe red (RR) stages, as well BP point stage treated with nitric oxide (BP2 + NO) and without NO (BP2-NO) (PRJNA668052). Up- and down-regulated genes are indicated in red and green colors, respectively, according to \(Z-\)score values obtained from 4-5 biological replicates. Statistical significance is shown in Table S5.