## 3 Discussion  
Asorbic acid (AsA, ascorbate, vitamin C) is well known for its indispensable roles in plant and human metabolism, primarily acting as an antioxidant, free radical scavenger, and cofactor for numerous enzymes [2]. However, humans cannot synthesize AsA. Thus, obtaining it from plant-based foods or pharmaceutical supplements is essential. In plants, AsA protects chloroplasts against ROS, is involved in ethylene synthesis, and acts as a  
Figure 10: Mass fraction of AsA in _G. max_ organs across developmental stages and under abiotic stress conditions: (**A**) seed germination; (**B**) leaf development; (**C**) roots under PEG and salinity stress; (**D**) leaves under PEG and salinity stress. Bar charts represent mean \(\pm\) standard deviation (\(n\) = 3). Different letters indicate significant differences (\(p\) < 0.05) between germination/development time points determined by the Bonferroni test. Asterisks indicate significant differences (\(p\) < 0.05) between the control and stress conditions determined by the t-test for each mass fraction. PEG stands for polyethylene glycol, Asc stands for ascorbate, and DM stands for dry matter. cofactor of dioxygenases, which are associated with abscisic acid (AsA) and gibberellin synthesis besides auxin catabolism [1; 2; 39]. Consequently, this direct relationship between AsA and phytohormones denotes the critical involvement of AsA during all phases of plant growth and development, in addition to abiotic and biotic plant responses. Therefore, studies on AsA biosynthesis regulation in plants can benefit crop improvement that promotes healthy plants with high yield stability, as well as the development of plants with higher levels of AsA. This study aimed to gain insight into the evolution and regulation of AsA biosynthesis genes under both development stages and stress conditions to identify genes that could be potentially advantageous for biotechnological application, followed by AsA-level quantification to cross-validate the expression analyses of the identified genes. The key findings of this study are illustrated in Figure 11. In addition, this study advances the concept of the connection between AsA biosynthesis and ROS scavenger activity of the AsA-GSH cycle [10]. Figure 11: Schematic representation of the main findings of this study: on the left, highly identical gene pairs and duplicated genes of _G. max_ compared with those of _Arabidopsis thaliana_ were identified in genomic analysis. The up- and downregulated genes involved in the four ascorbic biosynthesis pathways in _G. max_ during developmental and stress conditions are shaded in yellow and blue, respectively. Ascorbic acid quantification consistently validated the gene expression analyses. Genes marked with asterisks are highly stress-induced in stress-resistant genotypes. The genome-wide analyses identified 41 genes encoding the key enzymes (GMP, GGP, GPP, GalDH, GalLDH, GME, GulLO, GalUR, and MIOX) potentially involved in the four AsA biosynthesis pathways in _G. max_ against the total of 26 in _Arabidopsis_ (Figure 2, Supplementary Table S1). This higher gene number in _G. max_ seemed to be due to duplication events (Figure 2). Among these genes, some paralogous pairs revealed high nucleotide sequence identities between them, such as _Gm_GMP_1a_ and _1b_ (94.93%), _Gm_GMP_alpha-A_ and \(B\) (95.84%), _Gm_GMP_2a and _2b_ (96.99%), _GME_1a_ and _1b_ (91.38%), _GME_2a_ and _2b_ (93.15%), _Gm_GallDH_1a_ and _1b_ (93.31%), and _Gm_MIOX_1a_ and _1b_ (93.58%), indicating genes derived from recent duplication events as depicted in the phylogenetic trees (Figure 2). Studies reported duplicated genes in _G. max_ for other proteins such as EF1\(\alpha\)[40], PTOX [41], and the AsA-GSH cycle [10]. In effect, duplicated genes are widespread in the soybean genome, covering around 75% of genes [42].