Nitric oxide (NO) is a potent intercellular mediator of melanogenesis, whereas metallothionein (MT) is an inducible intracellular antioxidant that has been reported to scavenge NO. We investigated the existence and induction of MT in melanocytes, and its inhibitory effect on NO-induced melanogenesis. The expression of MT was detected in melanocytes, however, at a lower level than in keratinocytes, and its induction was possible by the addition of zinc chloride. Further, an NO-stimulated increase of tyrosinase activity in melanocytes was remarkably suppressed, when MT was induced prior to NO stimulation. Melanogenesis was also suppressed, when dexamethasone was used to induce MT. However, an NO-stimulated increase of tyrosinase expression was not suppressed at the gene and protein level, when MT was induced in melanocytes. The same suppressive effect of melanogenesis was also observed, when alpha-melanocyte-stimulating hormone or endothelin-1 was used as a stimulator. Because these results implied a mechanism other than NO scavenging to explain the suppressive effect of MT induction on melanogenesis, the direct inhibition of tyrosinase by MT was examined. Melanosome fractions were prepared from melanocytes, whose melanogenesis was suppressed by the induction of MT. Tyrosinase suppression was observed in the melanosome fractions, which was neutralized by the addition of anti-MT antibody. These results suggest that MT induction may be effective to suppress melanogenesis stimulated by NO as well as other melanogens, and these suppressive effects might be due to a direct inhibition of tyrosinase activity in melanosome and not a scavenging effect of NO.

Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene—an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive–negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T0 generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.this study was to confirm the relationship between alkaline phosphatase (ALP) and polyP metabolism in Saccharomyces cerevisiae. Our previous study suggests that a correlation exists between the amount of polyP and detectable ALP activity in arbuscular mycorrhizal (AM) fungi (Funamoto et al., 2007), which are obligate symbiotic microorganisms belonging to Glomeromycota (Schüßler et al., 2001) and which form associations with plant roots in a host nonspecific manner.AM fungi absorb Pi from the soil via extraradical hyphae. Absorbed Pi is then converted to polyP and translocates to arbuscules formed in the cortical cells of the plant root (Smith and Read, 1996). Enzymatic-histochemical experiments showed that ALP activity were detected in arbuscules (Ezawa et al., 1995;Gianinazzi et al., 1979;Tisserant et al., 1993). Additionally, it has been speculated that hydrolyzation of polyP occurs in arbuscules. ALP has occasionally been considered to be involved in polyP metabolism. However, analysis using the ALP specific inhibitor, Be 2+ , suggests that ALP of AM fungi has a high substrate specificity for sugar-phosphates, such as glucose-6-Pi and trehalose-6-Pi (Ezawa et al., 1999). Previously, our studies have demonstrated that ALP activity was high in arbuscules and polyP accumulation was low (Funamoto et al., 2007). Furthermore, when the expression of the AM-inducible Pi transporter gene of host plants was suppressed, the expression of AM ALP gene (GiALP) (Aono et al., 2004), which bears a high similarity to the Pi-deficient-induced type ALP gene (PHO8) of S. cerevisiae, was suppressed, and the ALP activity decreased and polyP accumulated in mature arbuscules (Funamoto et al., 2007). These results point to the hypothesis that ALP may play some role in polyP metabolism in arbuscules. However, it is difficult to obtain direct evidence to verify this hypothesis because conventional molecular biology methods, such as transformation and gene disruption, cannot be used in the case of AM fungi.

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