The D-mannose/L-galactose pathway plays a predominant role in ascorbate biosynthesis in the liverwortMarchantia polymorphabut is not regulated by light and oxidative stress

SUMMARY Ascorbate plays an indispensable role in plants, functioning as both an antioxidant and a cellular redox buffer. It is widely acknowledged that the ascorbate biosynthesis in the photosynthetic tissues of land plants is governed by light-mediated regulation of the D-mannose/L-galactose (D-Man/L-Gal) pathway. At the core of this light-dependent regulation lies the VTC2 gene, encoding the rate-limiting enzyme GDP-L-Gal phosphorylase. The VTC2 expression is regulated by signals via the photosynthetic electron transport system. In this study, we directed our attention to the liverwort Marchantia polymorpha , representing one of the basal land plants, enabling us to conduct an in-depth analysis of its ascorbate biosynthesis. The M. polymorpha genome harbors a solitary gene for each enzyme involved in the D-Man/L-Gal pathway, including VTC2 , along with three lactonase orthologs, which may be involved in the alternative ascorbate biosynthesis pathway. Through supplementation experiments with potential precursors, we observed that only L-Gal exhibited effectiveness in ascorbate biosynthesis. Furthermore, the generation of VTC2 -deficient mutants through genome editing unveiled the inability of thallus regeneration in the absence of L-Gal supplementation, thereby revealing the importance of the D-Man/L-Gal pathway in ascorbate biosynthesis within M. polymorpha . Interestingly, gene expression analyses unveiled a distinct characteristic of M. polymorpha , where none of the genes associated with the D-Man/L-Gal pathway, including VTC2 , showed upregulation in response to light, unlike other known land plants. This study sheds light on the exceptional nature of M. polymorpha as a land plant that has evolved distinctive mechanisms concerning ascorbate biosynthesis and its regulation.