Expression analysis of α-gliadin isoforms in wheat grains.

Gliadin is a major wheat seed storage protein that affects the extensibility of flour dough. Multiple genes encode gliadin, and there are numerous isoforms encoded by these genes, some of which might be related to flour quality. In this study, gliadin isoforms encoded by 30 α-gliadin genes from the wheat cultivar "Chinese Spring" (CS) were identified using 2-DE and MS/MS. The chromosomes where these isoform genes are located were determined using Gli-2 locus-deficient lines. A quantitative analysis by 2-DE revealed differences in expression levels among α-gliadin isoforms. We also separated the polymer and monomer fractions of the total protein by SEC. We found that an α-gliadin isoform with 7 cysteine residues was present at relatively higher levels in the polymer fraction than an α-gliadin isoform with 6 cysteine residues. The present study results can help in understanding the relationship between the properties of α-gliadin isoforms and the physical properties of dough in the future. SIGNIFICANCE: For investigating the relationship between isoforms and dough extensibility, we identified α-gliadin isoforms encoded by 30 genes among the 50 genes cloned until date. Moreover, the polymer and monomer fractions of the total protein were separated by SEC. We found that an α-gliadin isoform with 7 cysteine residues was present at relatively higher levels in the polymer fraction than an α-gliadin isoform with 6 cysteine residues. This study provided useful information for elucidating the relationship between the properties of α-gliadin isoforms and the physical properties of dough.

Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants.

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.

PHO8 gene coding alkaline phosphatase of Saccharomyces cerevisiae is involved in polyphosphate metabolism.

It has been argued for a long time whether alkaline phosphatase (ALP) is involved in polyphosphate (polyP) metabolism in arbuscular mycorrhizal fungi. In the present study, we have analyzed the effects of disrupting the PHO8 gene, which encodes phosphate (Pi)-deficiency-inducible ALP, on the polyP contents of Saccharomyces cerevisiae. The polyP content of the Δpho8 mutant was higher than the wild type strain in the logarithmic phase under Pi-sufficient conditions. On the contrary, the chain length of polyP extracted from the Δpho8 mutant did not differ from the wild type strain. When cells in Pi-deficient conditions were supplemented with Pi, the increase of the polyP amounts in the Δpho8 mutant was similar to that in the wild type strain. These results suggest that ALP, which is encoded by PHO8, affects the polyP content, but not the chain length, and participates in polyP homeostasis in Pi-sufficient conditions.