Marie-Hélène Morel

Reversible changes of the wheat γ46 gliadin conformation submitted to high pressures and temperatures

The structure of the wheat gamma 46 gliadin was investigated, in aqueous solutions, under high pressure or temperature by the use of ultraviolet and fluorescence spectroscopic techniques. We found that high pressure (above 400 MPa) induces a change in the protein conformation that results in a decrease of the polarity of the environment of aromatic amino acids. This new conformation was able to bind the hydrophobic probe, 8-anilino-1-naphtalene-sulfonic acid (ANS), indicating an increase in the gliadin surface hydrophobicity. Thermodynamic parameters of this conformational change were measured and infrared spectroscopy studies were used to probe the potential secondary structure modifications. The high stability of gamma 46 gliadin could be related to its elastic character, as the observed changes were always found to be reversible.

Characterization and quantification of low molecular weight glutenins in durum wheats.

Durum wheat proteins have been considered as a model because of the very clear-cut relationship previously evidenced between the electrophoretic type 42 or 45 of the components that are coded by the Gli-B1 chromosome locus and the intrinsic quality (gluten viscoelasticity) of cultivars. The proteins from 4 cultivars were subjected to sequential extraction and separated into five groups, respectively, in: NaCl, EtOH (gliadins-I), EtOH + mercaptoethanol (ME) (gliadins-II), AcOH + ME (glutenins-I) and SDS + ME (glutenins-II) and characterized using polyacrylamide gel electrophoresis (PAGE), SDS-PAGE and 2-dimensional (NEPHGE X SDS - PAGE) electrophoretic systems. EtOH-soluble fractions were also separated by ion-exchange chromatography, each fraction being characterized in PAGE and SDS-PAGE and its composition in major bands determined by densitometry. From the ratio of each chromatographic fraction and of each solubility group, an estimation of the major bands or electrophoretic zones was also made in respect to the whole proteins. In type 45 cultivars, it was shown that only 67% of the EtOH-soluble fraction (although considered as classical gliadins) had a monomeric character, giving rise to discrete bands in PAGE systems. The remainder (33%) were aggregated fractions, essentially those referred to as low molecular weight glutenins (LMWG), that migrate, upon reduction only, in SDS-PAGE systems. LMWG make up 27% of total proteins and are revealed as a strong triplet in the 44,500-51,500 MW region, in gliadin-I and especially in gliadin-II groups. In type 42 cultivars, the LMWG ratio is reduced about by half (18% of EtOH soluble fraction, 14% of total proteins). This difference, coupled with their aggregative behavior, leads to their consideration as the major functional markers of gluten quality, gliadins 42/45 being genetic markers only. Without excluding possible physicochemical differences between different LMWG allelic types, it is hypothesized that quantitative differences could explain by themselves the quality differences between the two durum wheat genetic types. Concerning the other aggregative fractions, like high molecular weight glutenin (HMWG) subunits in glutenin-I and II groups, they do not show (unlike bread wheats) quantitative or qualitative differences large enough to play a major role in explaining genetic differences in durum wheat gluten characteristics. It is recommended, especially for physicochemical studies of wheat quality, to rely on a protein classification based on monomeric or aggregative characteristics, instead of Osbornes scheme based only on fractionation by solubility.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic analysis of low Mr glutenin subunits fractionated by two-dimensional electrophoresis (A-PAGE × SDS-PAGE)

Alkylated glutenin subunits of F7 progenies from the cross between the Italian bread wheat cultivar Costantino and the Canadian cultivar Neepawa were fractionated by one-dimensional A-PAGE and SDS-PAGE and by two-dimensional A-PAGE × SDS-PAGE. Each gliadin allele at the Gli-1 loci of the parental cultivars was shown to be associated with a specific allele at each of the Glu-3 loci, at which low Mr glutenin subunits are encoded. The Glu-A3 locus was found to code for two low Mr subunits in Neepawa and three in Costantino. In this latter cultivar, eight low Mr subunits were assigned to each of the Glu-B3 and Glu-D3 loci, whereas seven subunits were attributed to the Glu-B3 locus and seven to the Glu-D3 locus in Neepawa. A-PAGE × SDS-PAGE can be employed for a detailed description of low Mr subunits of glutenin in different cultivars following a genetic approach based on the correspondence between the alleles at the Gli-1 and Glu-3 loci.

Responses to Hypoxia and Endoplasmic Reticulum Stress Discriminate the Development of Vitreous and Floury Endosperms of Conventional Maize (Zea mays) Inbred Lines

Major nutritional and agronomical issues relating to maize (Zea mays) grains depend on the vitreousness/hardness of its endosperm. To identify the corresponding molecular and cellular mechanisms, most studies have been conducted on opaque/floury mutants, and recently on Quality Protein Maize, a reversion of an opaque2 mutation by modifier genes. These mutant lines are far from conventional maize crops. Therefore, a dent and a flint inbred line were chosen for analysis of the transcriptome, amino acid, and sugar metabolites of developing central and peripheral endosperm that is, the forthcoming floury and vitreous regions of mature seeds, respectively. The results suggested that the formation of endosperm vitreousness is clearly associated with significant differences in the responses of the endosperm to hypoxia and endoplasmic reticulum stress. This occurs through a coordinated regulation of energy metabolism and storage protein (i.e., zein) biosynthesis during the grain-filling period. Indeed, genes involved in the glycolysis and tricarboxylic acid cycle are up-regulated in the periphery, while genes involved in alanine, sorbitol, and fermentative metabolisms are up-regulated in the endosperm center. This spatial metabolic regulation allows the production of ATP needed for the significant zein synthesis that occurs at the endosperm periphery; this finding agrees with the zein-decreasing gradient previously observed from the sub-aleurone layer to the endosperm center. The massive synthesis of proteins transiting through endoplasmic reticulum elicits the unfolded protein responses, as indicated by the splicing of bZip60 transcription factor. This splicing is relatively higher at the center of the endosperm than at its periphery. The biological responses associated with this developmental stress, which control the starch/protein balance, leading ultimately to the formation of the vitreous and floury regions of mature endosperm, are discussed.