Tissue- and Condition-Specific Biosynthesis of Ascorbic Acid in Glycine max L.: Insights from Genome-Wide Analyses of Pathway-Encoding Genes, Expression Profiling, and Mass Fraction Determination

Ascorbic acid (AsA) is an essential plant metabolite that acts primarily as an antioxidant, regulates cell division and elongation, and enhances stress tolerance. Despite its crucial physiological role, the biosynthesis of AsA in G. max, a major crop of significant commercial importance, remains largely unexplored. This gap highlights the need for a thorough investigation of AsA biosynthesis pathways and their role in optimizing the nutritional value and stress tolerance of soybeans. This study identified 41 key genes linked to four AsA biosynthesis pathways in G. max, highlighting specific gene duplications compared to Arabidopsis. Their expression levels were assessed by analyzing a diverse set of RNA-Seq data from the NCBI database. Additionally, to cross-validate the expression levels of genes and the accumulation levels of AsA in the principal tissues, G. max plants were grown under controlled conditions following the protocols from selected RNA-seq experiments. Genes associated with the D-mannose/L-galactose pathway exhibited ubiquitous expression, and the expression patterns of genes from alternative pathways reflected their responsiveness to specific tissues or environmental conditions. Germination and leaf development were accompanied by strong expression of gene members from all pathways, whereas leaf aging was characterized by downregulation. Specific gene members, such as GMP_2a (D-mannose/L-galactose pathway), GulLO_1f (L-gulose pathway), and MIOX_3a (Myo-inositol pathway) were highly stress-responsive and linked to stress-resistant genotypes and cultivars. Consistent with gene expression analyses, the quantification of AsA revealed the highest mass fractions in young leaves and germinating seeds. However, AsA mass fractions were significantly reduced or unchanged under stress conditions, depending on the type of stress and the duration of exposure. Overall, this study validated the relevance of AsA biosynthesis pathways in soybeans, highlighting key genes that could be targeted to enhance stress tolerance and improve ascorbate production, thereby boosting the nutritional value of soybeans.