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 (Dal1) showed 1.2fold 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 (Dvtc2-1) or PpVTC2-2 (Dvtc2-2), 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.

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Currently, there are limited reports available regarding the treatment and prevention of progressive age-related hearing loss. This is because age-related hearing loss is not a critical disease with direct fatalities and has several well-established countermeasures such as hearing aids and cochlear implants. This study evaluated the efficacy of Ninjinyoeito (NYT) in the treatment of age-related hearing loss. C57BL/6J mice were divided into three groups: baseline group, untreated group, and NYT-treated group, with the latter receiving NYT treatment for 2 months. The mice were fed with NYT extract mixed with 4% mouse normal chow. Hearing loss was confirmed by a reduction in intact cell density of the auditory nerve from the age of 5–7 months. The suppression of hearing loss with aging and decrease in the intact cell density of the auditory nerve were significant in mice fed with NYT for 2 months. NYT has been reported to improve blood flow and enhance mitochondrial activity and may exert its protective effects on spiral neurons through these mechanisms. There was no decrease in the size of the stria vascularis from the age of 5–7 months in C57BL/6J mice. The present model failed to reveal the effect of NYT on atrophy of the stria vascularis of the cochlear duct. In conclusion, NYT appears to have a protective effect on the auditory nerve and suppress the progression of age-related hearing loss by reducing age-related auditory nerve degeneration.