Pasting properties of A- and B-type wheat starch granules and annealed starches in relation to swelling and solubility.

Wheat starch contains two distinct groups of granules, A-type and B-type, which have different compositions and properties. The aim of this study was to investigate the differences in pasting properties of A- and B-type wheat starch granules and their annealed starches, and to relate them to swelling properties and solubility. A- and B-type wheat starch granules were fractionated. The differences in pasting properties between A- and B-type wheat starch granules depended on starch solids content. The A-type starch had a higher pasting viscosity at ≥8 % solids content, but the trend was reversed at a lower solids content (5 %). This cross-over phenomenon in the pasting viscosity can be explained because A-type wheat starch granules have more starch molecules leached out, while swelled less at high temperatures and are probably more rigid than B-type wheat starch granules. This is the first study to show the cross-over in the pasting viscosity-starch concentration between A-type and B-type wheat starches and that B-type wheat starch has higher pasting viscosity than A-type at a low solids content. When annealed in warm water, both annealed A- and B-type wheat starch granules had higher pasting viscosities than untreated counterparts by altering the swelling of starch.

Dynamic changes in ferulic acid and diferulic acids in wheat flour doughs during the breadmaking process.

Ferulic acid (FA), a phytochemical concentrated in wheat bran, influences structural characteristics of arabinoxylan (AX) and rheological properties of wheat dough. This study investigates the dynamic changes in FA and diferulic acids, closely associated with AX molecular weight, during the breadmaking process. FA predominantly exists in a tightly bound state within the arabinoxylan matrix, with a substantial increase in free FA content observed during the initial fermentation phase. Furthermore, this research identified four specific wheat-derived diferulic acids: 8-5'-DFA, 5-5'-DFA, 8-O-4'-DFA, and 8-5'-DFA (benzofuran form), tracking their variations throughout breadmaking. The notable upsurge in diferulic acid levels in the early fermentation stages suggests that the cleavage of ferulic acid moieties may not be the primary factor contributing to the reduction in AX molecular weight. Future investigations into the effects of FA and diferulic acids on arabinoxylan and wheat dough properties promise to enhance understanding of the intricacies of the breadmaking process.

Genetic Background Negates Improvements in Rice Flour Characteristics and Food Processing Properties Caused by a Mutant Allele of the PDIL1-1 Seed Storage Protein Gene.

Phenotypic differences among breeding lines that introduce the same superior gene allele can be a barrier to effective development of cultivars with desirable traits in some crop species. For example, a deficient mutation of the Protein Disulfide Isomerase Like 1-1 (PDIL1-1) gene can cause accumulation of glutelin seed storage protein precursors in rice endosperm, and improves rice flour characteristics and food processing properties. However, the gene must be expressed to be useful. A deficient mutant allele of PDIL1-1 was introduced into two rice cultivars with different genetic backgrounds (Koshihikari and Oonari). The grain components, agronomic traits, and rice flour and food processing properties of the resulting lines were evaluated. The two breeding lines had similar seed storage protein accumulation, amylose content, and low-molecular-weight metabolites. However, only the Koshihikari breeding line had high flour quality and was highly suitable for rice bread, noodles, and sponge cake, evidence of the formation of high-molecular-weight protein complexes in the endosperm. Transcriptome analysis revealed that mRNA levels of fourteen PDI, Ero1, and BiP genes were increased in the Koshihikari breeding line, whereas this change was not observed in the Oonari breeding line. We elucidated part of the molecular basis of the phenotypic differences between two breeding lines possessing the same mutant allele in different genetic backgrounds. The results suggest that certain genetic backgrounds can negate the beneficial effect of the PDIL1-1 mutant allele. Better understanding of the molecular basis for such interactions may accelerate future breeding of novel rice cultivars to meet the strong demand for gluten-free foods.

Analysis of molecular weight and structural changes in water-extractable arabinoxylans during the breadmaking process.

Arabinoxylans are important for dough and breadmaking properties. It is not clear how arabinoxylans of different molecular weights behave during the breadmaking process as well as the changes in individual structures. We investigated changes in the molecular weight and structure of water-extractable arabinoxylans. It was revealed that molecules larger than high molecular weight arabinoxylans were formed during the mixing and 1st fermentation (105 min before 1st punch). High molecular weight arabinoxylan continued to be degraded from mixing to the proofing stage. The arabinose to xylose ratio increased at mixing and the 1st fermentation due to solubilization of highly substituted arabinoxylan. Low molecular weight arabinoxylan did not show degradation and structural changes during the fermentation process, whereas the weight average molecular weight of low molecular weight arabinoxylan significantly decreased (P < 0.05) at mixing. Water extractable arabinoxylan shows different behaviors for molecular weight and structural changes during the breadmaking process.

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.

Changes in Protein Non-Covalent Bonds and Aggregate Size during Dough Formation.

This research investigated changes in the amounts and sizes of monomeric proteins and protein aggregates during dough mixing, with a focus on the contribution of non-covalent bonds in the aggregation of gluten proteins. High protein flour (HF) and low protein flour (LF) were used in this study. As dough mixing progressed from flour to overmixed dough, the total amount of protein aggregates increased while the amount of monomeric protein decreased. Omega-gliadin was the major monomeric protein that decreased in quantity. Interestingly, the amount of larger-sized protein aggregates decreased and that of smaller-sized protein aggregates increased. The amount of gluten protein macro-polymer aggregated through strong non-covalent bonds decreased whereas aggregates formed with weaker non-covalent bonds increased. LF dough behaved similar to HF dough. Large-sized gluten protein aggregates disaggregated due to the weakening of non-covalent bonds and became smaller. Omega-gliadin was incorporated into gluten protein aggregates during dough mixing.

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.

Comprehensive molecular characterization of the α/ß-gliadin multigene family in hexaploid wheat.

To characterize the structure and expression of a large multigene family of α/ß-gliadin genes, 90 individual α/ß-gliadin genes harboring a promoter region were identified in the wheat cultivar Chinese Spring. These genes were classified into eleven groups by phylogenetic analysis, and the chromosomes they were derived from were determined. Of these genes, 50 had the basic α/ß-gliadin domains and six conserved cysteine residues and 16, 16 and 18 of them were, respectively, located on chromosome 6A, 6B and 6D. Six genes had an additional cysteine residue, suggesting that these α/ß-gliadins acquired the property of binding other proteins through intermolecular disulphide bands. Expression of α/ß-gliadin genes in developing seeds was measured by quantitative RT-PCR using group-specific primers over 3 years. Expression patterns of these genes on the basis of accumulated temperature were similar among gene groups, whereas expression levels differed for the 3 years. The expression of most α/ß-gliadin and other prolamin genes was correlated with the sunshine duration. On the other hand, although all α/ß-gliadin genes had a common E-box within the -300 promoter region, some genes showed a particular expression pattern with respect to the sunshine duration, similarly to gene encoding high-molecular weight glutenin subunits and endosperm enzymes. These observations suggested that expression of each α/ß-gliadin gene is differentially regulated by multiple regulatory factors.

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B.

BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Oral administration of Lactobacillus plantarum strain AYA enhances IgA secretion and provides survival protection against influenza virus infection in mice.

The mucosal immune system provides the first line of defense against inhaled and ingested pathogenic microbacteria and viruses. This defense system, to a large extent, is mediated by the actions of secretory IgA. In this study, we screened 140 strains of lactic acid bacteria for induction of IgA production by murine Peyer's patch cells. We selected one strain and named it Lactobacillus plantarum AYA. We found that L. plantarum AYA-induced production of IL-6 in Peyer's patch dendritic cells, with this production promoting IgA(+) B cells to differentiate into IgA-secreting plasma cells. We also observed that oral administration of L. plantarum AYA in mice caused an increase in IgA production in the small intestine and lung. This production of IgA correlated strongly with protective ability, with the treated mice surviving longer than the control mice after lethal influenza virus infection. Our data therefore reveals a novel immunoregulatory role of the L. plantarum AYA strain which enhances mucosal IgA production and provides protection against respiratory influenza virus infection.

Next-generation survey sequencing and the molecular organization of wheat chromosome 6B.

Common wheat (Triticum aestivum L.) is one of the most important cereals in the world. To improve wheat quality and productivity, the genomic sequence of wheat must be determined. The large genome size (∼17 Gb/1 C) and the hexaploid status of wheat have hampered the genome sequencing of wheat. However, flow sorting of individual chromosomes has allowed us to purify and separately shotgun-sequence a pair of telocentric chromosomes. Here, we describe a result from the survey sequencing of wheat chromosome 6B (914 Mb/1 C) using massively parallel 454 pyrosequencing. From the 4.94 and 5.51 Gb shotgun sequence data from the two chromosome arms of 6BS and 6BL, 235 and 273 Mb sequences were assembled to cover ∼55.6 and 54.9% of the total genomic regions, respectively. Repetitive sequences composed 77 and 86% of the assembled sequences on 6BS and 6BL, respectively. Within the assembled sequences, we predicted a total of 4798 non-repetitive gene loci with the evidence of expression from the wheat transcriptome data. The numbers and chromosomal distribution patterns of the genes for tRNAs and microRNAs in wheat 6B were investigated, and the results suggested a significant involvement of DNA transposon diffusion in the evolution of these non-protein-coding RNA genes. A comparative analysis of the genomic sequences of wheat 6B and monocot plants clearly indicated the evolutionary conservation of gene contents.

Development of taxon-specific sequences of common wheat for the detection of genetically modified wheat.

Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.