Glutelin subtype-dependent protein localization in rice grain evidenced by immunodetection analyses.

KEY MESSAGE: GluA and GluB-4/5 glutelin subfamilies are mainly localized to outer region of the endosperm, particularly in its ventral side, in rice grain, but GluC is localized to throughout the endosperm. The major seed storage protein in rice (Oryza sativa) is glutelin, which forms a vacuole-derived protein body type-II. Glutelins are encoded by multiple genes, and generally comprise four protein subfamilies, namely, GluA, GluB, GluC, and GluD: however, the localization pattern of glutelin in rice grains remains obscure. In this study, we investigated the localization pattern of five subtypes of the glutelin protein in rice grains using glutelin-subtype specific antibodies. Immunoblot analysis against sequentially polished rice flour fractions from three crop years and seven japonica rice varieties revealed that GluA was strongly localized in the outer region of the endosperm, including the subaleurone layer, whereas GluC was distributed throughout the endosperm. Among the glutelin subtypes, GluA was mostly found in the outer region of the rice grain, followed by GluB-4/5, GluB-1, GluD, and GluC. Immunofluorescence labeling microscopy analysis using immature rice seeds clearly revealed that the localization pattern of GluC and GluD was completely different from that of GluA and GluB. Expression levels of all glutelins, particularly GluA, GluB-1, and GluB-4/5, were stronger on the ventral than dorsal side in rice grains. These results provide strong and consistent evidence that glutelins localize to the rice grain in a subfamily-dependent manner.

Cloning and characterization of a novel low-molecular-weight glutenin subunit gene with an unusual molecular structure of Aegilops uniaristata.

Low-molecular-weight glutenin subunits (LMW-GSs) are one of the important factors for the dough processing quality. In this study, a novel LMW-GS, designated LMW-N13, from the wheat relative species Aegilops uniaristata PI 554421 was cloned and characterized. Unlike previously published LMW-GSs, LMW-N13 has a large molecular weight and is the largest LMW-GS published thus far. Sequence alignments demonstrated that LMW-N13 is a LMW-i-type subunit but contains nine cysteine residues which is one more than typical LMW-i-type subunits. In addition, four insertions are present in the repetitive domain that resulted in the large molecular weight. In vitro analysis showed that LMW-N13 could improve the dough quality of different base flours.

Characterization of a novel glutelin subunit OsGluBX by the experimental approach and molecular dynamics simulations.

Rice glutelin is a multi-subunit storage protein and has high nutritional value. However, many glutelin subunits are still not identified by experiment approach. In this study, a novel subunit (OsGluBX) was discovered by sequence alignment in the UniProtKB database. And then, the OsGluBX of rice from japonica cv. Nipponbare and indica cv. 9311 were cloned and expressed in Escherichia coli system and further identified by Western blotting. The total storage proteins were extracted from the grains of Nipponbare and 9311, and the native OsGluBX were identified. The novel OsGluBX subunit was classified into the subfamily B based on its high homology to the subfamily B members and their immunoblotting identification against the subfamily-specific antibody. Furthermore, two-dimensional electrophoresis analysis showed similarity and difference of the entire glutelin profiles between the two subspecies. Moreover, the atomic coordinate of the OsGluBX was constructed based on homology modeling approach and refined by molecular dynamics simulations. The spatial conformation of the OsGluBX protein was stable during the simulation, and the obvious hydrogen bonds were observed to maintain the integrity and stability of the ß-sheets region of the OsGluBX. Research into this novel OsGluBX subunit has improved our understanding of the glutelin family in rice.

Polymorphism of the storage proteins in Portuguese rye (Secale cereale L.) populations.

Currently, due to the abandonment of traditional agricultural practices and the decline of rye production in Portugal, there is a need to assess the genetic diversity of rye in order to preserve its biodiversity. Furthermore, a greater knowledge of rye secalins is important for rye bread-making quality and other crop breeding purposes. The genetic variation and diversity of storage proteins were estimated for fourteen populations of rye (Secale cereale L.) sampled in northern Portugal. The work showed the high genetic diversity within the Portuguese rye gene-pool as an important source for plant breeding and emphasized the necessity of an integrated resources genetic program to allow a more efficient management and conservation of these resources. The rye populations were compared with 'Picasso' and 'Marder' varieties. Several alleles were identified by the single electrophoretic mobility patterns. We studied a set of 1600 rye seeds, including regional populations and varieties, having observed a total of 24, 5, 21 and 47 alleles for HMW, 75k γ-, 40k γ- and ω-secalins, respectively. The coefficient of similarity within populations is presented using cluster representation. The mean value of genetic variation indices (H) for rye storage proteins was very high in regional populations, ranging from 0.67 to 0.78, while in the varieties ranged from 0.57 to 0.58. Knowledge of the diversity of secalins will increase our understanding of the quality differences between rye varieties, especially considering the relative small number of rye cultivars grown around the world.

Phylogenetic analysis of C, M, N, and U genomes and their relationships with Triticum and other related genomes as revealed by LMW-GS genes at Glu-3 loci.

Phylogenetic relationships between the C, U, N, and M genomes of Aegilops species and the genomes of common wheat and other related species were investigated by using three types of low-molecular-weight glutenin subunit (LMW-GS) genes at Glu-3 loci. A total of 20 LMW-GS genes from Aegilops and Triticum species were isolated, including 11 LMW-m type and 9 LMW-i type genes. Particularly, four LMW-m type and three LMW-i type subunits encoded by the genes on the C, N, and U genomes possessed an extra cysteine residue at conserved positions, which could provide useful information for understanding phylogenetic relationships among Aegilops and Triticum genomes. Phylogenetic trees constructed by using either LMW-i or the combination of LMW-m and LMW-s, as well as analysis of all the three types of LMW-GS genes together, demonstrated that the C and U genomes were closely related to the A genome, whereas the N and M genomes were closely related to the D genome. Our results support previous findings that the A genome was derived from Triticum uratu, the B genome was from Aegilops speltoides, and the D genome was from Aegilops tauschii. In addition, phylogenetic relationships among different genomes analysed in this study support the concept that Aegilops is not monophyletic.

[Polymorphism of hordein-coding loci in barley (Hordeum vulgare L.) populations of Iran and Central Asian countries].

Starch gel electrophoresis was performed to study polymorphism of hordeins encoded by the Hrd A, Hrd B, and Hrd Floci in 366 local old barley accessions from Iran and Central Asian countries, including Turkmenistan, Uzbekistan, Tajikistan (Mountain Badahsan), and Kirgizia. In total, 60 alleles with frequencies of 0.0003-0.2818 were observed for the Hrd A locus, 106 alleles with frequencies of 0.0003-0.1603 were observed for the Hrd B locus, and five alleles with frequencies of 0.0164-0.4131 were observed for the Hrd Flocus. The alleles and allele frequencies displayed irregular distributions in barley populations of the above countries. Cluster analysis of the matrix of allele frequencies in populations from known collection sites revealed a cluster structure of local barley populations within each country. Local populations formed five differently sized clusters in Iran, six in Turkmenistan, three in Uzbekistan, and three in Kirgizia. The variation and allele frequency distribution of the hordein-coding loci in Iran and Central Asian countries were assumed to result from the introduction and spreading of barley forms via migrations of husbandmen.

Identification and characterization of high-molecular-weight secalins from triticale seeds by capillary zone electrophoresis.

A rapid and reliable method for separation and characterization of the variability of high-molecular-weight secalin subunits (HMW-SS) in hexaploid triticale (x Triticosecale Wittmack) by CZE has been developed. In this method, a mixture of two poly(ethylene oxide) polymers differing in molecular weight and a high concentration of ACN in isoelectric buffer was applied as the running electrolyte. For dynamic coating of the capillary inner wall, a low-concentration mixture of poly(vinylpyrrolidone) and hydroxypropylmethylcellulose was employed. Wide allelic variations in rye HMW-SS composition, including some novel x- and y-type HMW-SS, were detected by CZE. The CZE electropherograms of HMW-SS showed two groups of peaks in accordance with y- and x-type subunits, with migration times of 8.0-8.8 and 11.0-13.3 min, respectively. HMW-SS differed in migration times from the simultaneously resolved HMW glutenin subunits, but frequently had very similar electrophoretic mobilities during separation by SDS-PAGE. Each of the two rye subunits 2r and 6.5r detected by SDS-PAGE represents in fact two subunits (5.1r or 5.2r, and 6.4r or 6.5r, respectively). After analyzing 106 European triticale cultivars, 12 HMW-SS were identified (six x-type and six y-type). They form six allelic variants of these subunits. The simultaneous separation and identification of triticale HMW glutenin and secalin subunits by CZE is an efficient alternative to SDS-PAGE and should facilitate breeding of valuable cultivars.

Structural variation and evolutionary relationship of novel HMW glutenin subunits from Elymus glaucus.

High molecular weight (HMW) glutenin subunits (GS) are important seed storage proteins relevant to the end-use quality of wheat and other cereal crops. Here we report the isolation and characterization of two novel HMW-GS alleles (1St 1.4 and 1St1.1) from the perennial Triticeae species Elymus glaucus. The amino acid (aa) sequences of E. glaucus 1St1.4 and 1St1.1 were predicted as 434 aa and 358 aa, respectively. Both subunits comprise a signal peptide with a conserved N-terminal domain, a central repetitive domain and a C-terminal domain. Elymus glaucus 1St 1.4 and 1St1.1 exhibit several distinct characteristics different from other known HMW-GSs. The lengths of repetitive domains in E. glaucus 1St 1.4 and 1St1.1 are substantially smaller than those of other known HMW-GSs, in which 1St1.1 (only 358 aa) is the smallest subunit identified so far. The N-terminal domains of E. glaucus 1St 1.4 and 1St1.1 are homologous to y-type subunits, whereas their C-terminal domains are similar to x-type subunits. Our results indicate that E. glaucus 1St 1.4 and 1St1.1 are novel HMW-GS variants or isoforms, and the characterization of both subunits can enhance our understanding on the structural differentiation and evolutionary relationship of HMW-GSs in Triticeae species.

Enfermedad Celíaca: diagnostico y nuevas formas de presentación / Celiac disease: diagnosis and new forms of presentation

Es posible que la Enfermedad Celíaca (EC) en algunos países esté aún subdiagnosticada, y Colombia no es la excepción. Hay diversas formas de presentación. en cualquiera de los casos, es necesario las pruebas de anticuerpos y la toma de biopsia intestinal para su diagnóstico. El diagnóstico de EC, según lo recomendado desde el año 1989 por ESPGHAN, con la toma de unabiopsia intestinal ante la presencia de síntomas, para luego del retiro del gluten esperar reversiónde la sintomatología junto con seronegatividad de los anticuerpos.

Celiac Disease (CD) in some countries is still underdiagnosed, and Colombia is no exception. There are different forms. In either case, it is necessary antibody testing and intestinal biopsy for diagnosis. The diagnosis of CD, is checked as recommended since 1989 by ESPGHAN, with the capture of an intestinal biopsy in the presence of symptoms, then removal of gluten expected reversal of symptoms with seronegative for antibodies.

[Characterization of low-molecular-weighT i-type glutenin subunit genes from diploid wheat in relation to the gene family structure].

The low-molecular-weight (LMW) glutenin subunits are important for aspects of wheat quality and dough processing, and the LMW-i type glutenin is one of the typical glutenins. However, a detailed description of the DNA structure and encoded polypeptides of the LMW-i type glutenin subunit gene family is still lacking. In this study, two LMW-i type glutenin subunit genes, i.e., LMW-Eb from Triticum boeoticum and LMW-Em from T. monococcum, were obtained from genomic DNA, respectively. The LMW-Eb is a novel gene and the LMW-Em has the identical sequence with a known gene. To comprehensively understand the LMW-i type glutenin subunit gene family structure, all known LMW-i type glutenin subunit genes were comparatively analyzed. Detailed comparison of these genes revealed a high-level of single nucleotide polymorphisms (SNP). In these LMW glutenin subunits, the percentage of glutamine and proline were approximately 38.27 and 12.77%, respectively. They started directly from the repetitive domain with ISQQQ- after the signal sequence, which the N-terminal regions were absent. In addition, there are three consensus repeat motifs (i.e. PPFSQQQQ, PPISQQQQ and PPYSQQQQ) in the repetitive domains of these LMW glutenin subunits. The C-terminal I domain is the most conserved region, while the domains of C-terminal II and III are more variable. The eight cysteine residues are highly conserved. These genes could be clustered into two major groups, among which one group could be further divided into five subgroups. Furthermore, to date, all known LMW-i type glutenin subunit genes are located on chromosome 1A, whereas no LMW-i type glutenin subunit gene is obtained from the B and D genomes in wheat.

Evolution of the high molecular weight glutenin loci of the A, B, D, and G genomes of wheat.

We used PCR to obtain phylogenetically informative sequences from the high molecular weight glutenin genes of wheat. The validity of partial sequence comparisons as a means of studying glutenin phylogenetics was established by constructing neighbour-joining trees from partial alignments of 12 published glutenin allele sequences. PCR was then used to obtain 20 novel glutenin allele sequences from various Triticum and Aegilops species, including a 3000 year old preserved wheat. A neighbour-joining tree derived from all known glutenin allele sequences had eight clades, representing the eight loci from which the allele sequences were derived, and was split into two halves, one comprising alleles from the Glu-1-1 loci and the other comprising Glu-1-2 alleles. The topology was compatible with the postulated relationships between the A, B, D, and G genomes. The Glu gene duplication event was tentatively dated at 7.2-10.0 million years ago (MYA), the origin of the four genomes at 5.0-6.9 MYA, and the split between the B and G genomes at 2.5-3.5 MYA. The Glu-B1-1 alleles in cultivated wheats fell into two subgroups that diverged 1.4-2.0 MYA, suggesting that emmer was domesticated twice. The D allele sequences were relatively diverse, indicating that the hybridization event that resulted in the hexaploid bread wheats might have occurred more than once.