Integrated Multi-Omics Analysis Reveals Glycosylation Involving 2-O-β-D-Glucopyranosyl-L-Ascorbic Acid Biosynthesis in Lycium barbarum

L-ascorbic acid (vitamin C, AA) is widely present in plants, but humans lack the ability to synthesize it independently. As a potent reducing agent, AA is susceptible to oxidation, making the enhancement of its stability crucial. 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG) is a stable natural derivative of AA with glycosylation, initially discovered in the fruits of <i>Lycium barbarum</i>. Understanding the biosynthesis of AA-2βG is crucial for enhancing its production in <i>L. barbarum</i>. While the established biosynthesis pathway of AA constitutes the upstream of AA-2βG biosynthesis, the conclusive step of β-glycosylation remains unclear. We identified a <i>L. barbarum</i> cultivar by UPLC, ZN01, with a high content of AA-2βG, and compared its leaves, immature fruits, and mature fruits to a normal AA-2βG content <i>L. barbarum</i> cultivar for metabolomic and transcriptomic analysis. The RNA-seq and RT-qPCR analysis revealed that the expression levels of genes involved in the AA biosynthesis pathway did not consistently correlate with AA-2βG content, suggesting that the final glycosylation step may be a key determinant of AA-2βG accumulation. Subsequently, utilizing phylogenetic and co-expression analysis, we identified ten UDP-glycosyltransferases (UGTs) and three β-glucosidases (BGLUs) which may be involved in the crucial step of the conversion from AA to AA-2βG, and the UGTs' activities were predicted through molecular docking. Lastly, we speculated that the presence of the glycosylation process of AA might have a crucial role in maintaining AA homeostasis in <i>L. barbarum</i>, and deliberated on potential correlations between AA, carotenoids, and anthocyanins. Our integrated multi-omics analysis provides valuable insights into AA-2βG biosynthesis in <i>L. barbarum</i>, identifying thirteen candidate genes and highlighting the complex interplay between AA, carotenoids, and anthocyanins. These findings have implications for improving AA-2βG content in <i>L. barbarum</i>.