The d‐mannose/l‐galactose pathway is the dominant ascorbate biosynthetic route in the moss Physcomitrium patens. (10th August 2021)

• Record Type: Journal Article
• Authors: Sodeyama, Tsubasa; Nishikawa, Hitoshi; Harai, Kenji; Takeshima, Daiki; Sawa, Yoshihiro; Maruta, Takanori; Ishikawa, Takahiro
• Is Part Of: Plant journal. Volume 107:Number 6(2021)
• Page Start: 1724
• Page End: 1738
• Publication Date: 2021-08-10
• Subjects: ascorbate biosynthesis -- d‐mannose/l‐galactose pathway -- aldonolactonase -- Physcomitrella -- redox; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.15413
• Languages: English
• ISSNs: 0960-7412

Abstract

Summary: Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d ‐mannose/l ‐galactose (d ‐Man/l ‐Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d ‐galacturonate (d ‐GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d ‐Man/l ‐Gal pathway and an orthologous gene encoding aldonolactonase (ALase) – a key enzyme for the d ‐GalUA pathway – Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2 . Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l ‐galactonic acid to the final precursor l ‐galactono‐1, 4‐lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 ( Δal1 ) showed 1.2‐fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2‐1 ( Δvtc2‐1 ) or PpVTC2‐2 ( Δvtc2‐2 ), which encode the rate‐limiting enzyme GDP‐l ‐Gal phosphorylase in the d ‐Man/l ‐Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P . patens is the d ‐Man/l ‐Gal pathway, not the d ‐GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis. Significance Statement: The d ‐mannose/l ‐galactose (d ‐Man/l ‐Gal) pathway is the dominant l ‐ascorbate (ASC) biosynthetic pathway in vascular land plants and some green algae; however, an alternative pathway utilizing d ‐galacturonate (d ‐GalUA) has also been proposed. Physcomitrium patens possesses orthologs of genes encoding aldonolactonase (ALase), a key enzyme in the d ‐GalUA pathway, in addition to the complete set of genes encoding enzymes involved in the d ‐Man/l ‐Gal pathway. The current study shows that, in P. patens, the dominant pathway for ASC biosynthesis is the d ‐Man/l ‐Gal pathway, not the d ‐GalUA pathway, and ALase play a pivotal role in ASC metabolism via oxidized ascorbate degradation rather than ASC biosynthesis.