## 2 Results  
### Identification and Phylogenetic Relationship of Genes Encoding AsA Biosynthesis Pathways in Soybean  
Through BLASTn, using genes (cDNAs) that encode AsA enzymes in _Arabidopsis_ as a query, 41 orthologous sequences were identified in the soybean genome available in the GenBank database (Supplementary Table S1). The cDNAs and the corresponding proteins for each gene were retrieved from RefSeq mRNA and/or protein databases and subsequently used in gene expression and phylogenetic analyses. Phylogenetic trees were constructed by aligning cDNA sequences from \(G\). _max_ (41), _A. thaliana_ (26), and _Ambrella_ (23) (Figure 2, Supplementary Table S1), encoding the main proteins (GMP, GGP, GPP, GalDH, GalLDH, GME, GulLO, GalUR, and MIOX) involved in AsA synthesis. The protein sequences encoding the principal G. mannose/L. galactose pathway-encoding genes (GGP_1a, 1b, 1 LikeA, and 1 LikeB), GPP (GPP_L, 1 and 2), GalDH (GalDH_1a and 1b), GalLDH (GalLDH_1a and 1b), and GME (GME_1a, 1b, 2a, 2b)) were well clustered with their previously reported homologous genes in _A. thaliana_ (Figure 2a-f). GulLO (GuILO_1a, 1b, 1c, 1d, 1e, 1f, 1g, and 3) from the L-Gulose pathway (Figure 2g), the D-Galacturonate pathway (GalUR (GalUR_1, 2, 3, 4 and 5)) (Figure 2h), and the MIOX_1a, 1b, 2a, 2b, 3a, and 3b genes were clustered (Figure 2i), whereas the _Amborella_ sequences taken as an outgroup delineated the evolutionary relationship and highlight the distinct clustering of _G. _max_ and _A. thaliana_ genes to each distinct pathway. Except for GPP, in which _Arabidopsis_ and _G. _max_ comprise three gene members (Figure 2c), all other gene families showed a higher number of gene members in _G. _max_ than in _A. thaliana_. Gene Expression Analyses Indicate That All Proposed AsA Biosynthesis Pathways Play an Active Regulatory Role in _G. _max_ Species  
The substantial expression level of pathway-specific genes across tissues (flower, another, ovary, pod, leaf, root, root nodule, and seed) and in different physiological conditions (somatic embryogenesis, seed germination, and developmental stages) under ordinary environmental conditions evidences the active regulatory role of the four suggested AsA biosynthesis pathways in _G. _max_ (Figure 3). In Supplementary Table S3, the RPKM values reflect the expression levels of specific genes across tissues and conditions. Some gene members of the D-mannose/L-galactose pathway showed tissue-specific expression, such as _GGP_1a_ and _1b_ in the leaves and cotyledon and during somatic embryogenesis seed development; _GME_1a_ was observed to be well expressed during seed germination, seed development, and in cotyledons; in addition, _GGP_1-likeA_ was also well expressed in seed development. For the other pathways, a notable emphasis was observed on genes associated with the galacturonate pathway, particularly _GalUR_2_ and 7, in roots and during seed germination. The expression analyses indicated that gene families associated with the D-mannose/L-galactose pathway were consistently detected, with at least one gene member exhibiting Log2(RPKM) values above 1.2 (on a scale ranging from 0 to 2.5) in each tissue evaluated. The genes with the highest expression level from the pathways included _GMP_1a_ and _1b_, _GGP_1a_ and _1b_, _GPP_1, _GalLDH_1a_, _GalLDH_1a_, and _GME_1a_, _2a_, and _2b_. Notably, these genes were most highly expressed in flowers, pods, and leaves (Figure 3). Regarding the gene families associated with the other three pathways, they were less expressed and showed specific gene members linked to particular tissues. In the L-glucose pathway, the majority of genes were minimally detected. Among them, _GulLO_3_ was the most highly expressed gene and, together with _GulLO_1c_, _was_ ubiquitously expressed. However, _GulLO_1a_ was detected only in roots (including nodules); _GulLO_1d_ was expressed in flowers (including anthers and ovaries), leaves (V3), and roots; and _GulLO_1b_, _1e_, _1f_, and _1g_ were not detected in seeds.