Project description:Food processing conditions affect the structure, solubility and therefore accurate detection of gluten proteins. We investigated the influence of dough, bread and pretzel making on the composition of different wheat protein fractions obtained by Osborne fractionation, which is based on solubility. The albumin/globulin (ALGL), gliadin and glutenin fractions from flour, dough, crispbread, bread and pretzel were analysed using RP-HPLC, SDS-PAGE and untargeted nanoLC-MS/MS. This approach enabled an in-depth profiling of the fractionated proteomes and related compositional changes to processing conditions including mixing, heat and alkali treatment. The ALGL fractions contained 71 protein groups, the gliadin fractions 27 and the glutenin fractions 47, of which 16 were common in all fractions. Label-free quantitation revealed that the relative abundances of 78 proteins were significantly affected by baking in the fractions of bread crumb and bread crust in comparison to flour.
Project description:Frozen dough baking is useful method in the modern bread-making industry. However, the fermentation activity of baker’s yeast dramatically decreased after thawing due to freeze injuries, because baker’s yeast cells contained in dough experience freeze injuries during freeze-thaw processes. Here, we performed genome-wide expression analysis to determine genetic response in baker’s yeasts under freeze-thaw condition using a DNA microarray analysis. Functional and clustering analyses in gene expression reveal that genes could be characterized by the term of freeze-thaw stress. Under short-term freeze stress (freeze treatment for 3 day), genes involved in ribosomal protein were up-regulated. Under long-term freeze stress (freeze treatment for longer than 7 day), genes involved in energy synthesis were up-regulated. In each phase, genes involved in protein damage, several stresses and trehalose and glycogen metabolism were also up-regulated. Through these freeze stress, yeast cells may improve reduced efficiency of translation and enhanced cell protection mechanism to survive under freeze stress condition. These regulations of these genes would be controlled by the cAMP-protein kinase A pathway. Keywords: baker’s yeast, freeze-thaw stress, gene expression, freezing period
Project description:The high molecular weight (HMW) subunits of wheat glutenin are synthesised only in the starchy endosperm tissue of the developing wheat grain. We studied the effect of introducing transgenes on the global gene expression profiles of selected transgenic wheat lines, particularly during wheat seed development. For these particular set of experiments a direct comparison between the hexaploid bread transgenic line B102,1-1 (Rooke, L., Steele, S.H., Barcelo, P.,Shewry, P.R. & Lazzeri,P. Transgene inheritance, segregation and expression in bread wheat. Euphytica 129, 301-309 (2003)) and it background, non transformed L88-31 wheat line (Lawrence,G.J., Macritchie, F. & Wrigley, C.W. Dough and baking quality of wheat lines in glutenin subunits controlled by Glu-A1, Glu-B1 and Glu-D1 loci. J. Cereal. Sci. 7,109-112 (1988)) was performed. Transcriptome comparison analysis was performed in endosperm tissue (14 and 28 days post anthesis-dpa) and in leaf tissue (8 days post germination ?dpg). The transcriptome comparisons analysis was performed using three biological replicates (i.e. per line/tissue /developmental stage selected). Hybridisations were performed in reverse dye labelling.
Project description:Frozen dough baking is useful method in the modern bread-making industry. However, the fermentation activity of bakerâs yeast dramatically decreased after thawing due to freeze injuries, because bakerâs yeast cells contained in dough experience freeze injuries during freeze-thaw processes. Here, we performed genome-wide expression analysis to determine genetic response in bakerâs yeasts under freeze-thaw condition using a DNA microarray analysis. Functional and clustering analyses in gene expression reveal that genes could be characterized by the term of freeze-thaw stress. Under short-term freeze stress (freeze treatment for 3 day), genes involved in ribosomal protein were up-regulated. Under long-term freeze stress (freeze treatment for longer than 7 day), genes involved in energy synthesis were up-regulated. In each phase, genes involved in protein damage, several stresses and trehalose and glycogen metabolism were also up-regulated. Through these freeze stress, yeast cells may improve reduced efficiency of translation and enhanced cell protection mechanism to survive under freeze stress condition. These regulations of these genes would be controlled by the cAMP-protein kinase A pathway. Experiment Overall Design: All experiments were done in duplicate from two independent samples.
Project description:The high molecular weight (HMW) subunits of wheat glutenin are synthesised only in the starchy endosperm tissue of the developing wheat grain. We compared the expressed genomes of the transgenic wheat line B102,1-1 (Rooke et al. Transgene inheritance, segregation and expression in bread wheat. Euphytica 129, 301-309 (2003)). Both lines were shown to express the HMW-GS Ax1 gene (Halford, N.G. et al. Analysis of HMW glutenin subunits encoded bychromosome 1A of bread wheat (Triticum aestivum L.) indicates quantitative effects on grain quality. Theor Appl Genet 83, 373-378 (1992).) to the expressed genome of conventionally bred wheat line L88-18 (Lawrence et al. Dough and baking quality of wheat lines in glutenin subunits controlled by Glu-A1, Glu-B1 and Glu-D1 loci. J. Cereal. Sci. 7,109-112 (1988)) which results in the same effects on traits. Transcriptomes comparison analysis was performed in endosperm tissue (14 and 28 days post anthesis-dpa) and in leaf tissue (8 days post germination dpg), respectively. Each of the transcriptome comparisons was performed using three biological replicates (i.e. per line/tissue /developmental stage selected). Hybridisations were performed in reverse dye labelling.Exceptionally, biological replica 2 was only performed for B102,1-1 (green)/L88-18 (red) labelling and not swap
Project description:Drought stress is becoming more prevalent with global warming, and has been shown to have large effects on gluten proteins linked to wheat bread making quality. Likewise, low temperature stress can detrimentally affect proteins in wheat. This study was done to determine the differential expression of high molecular weight (HMW) glutenin proteins in a drought and low temperature stressed high quality hard red spring wheat cultivar (PAN3478), against a control. The treatments were applied in the greenhouse at the soft dough stage. HMW glutenin proteins were extracted from the flour, and separated by two-dimensional gel electrophoresis. Protein spots that had p values lower than 0.05 and fold value equal to or greater than 1.2 were considered significantly differentially expressed. These proteins were further analyzed by tandem mass spectrometry.
Project description:Waxy starch has an important influence on bread dough and the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. The waxy and non-waxy wheats had similar starch granule morphological features and developmental patterns, and similar amylopectin quality in the grain. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Dynamic transcriptional expression profiling of starch biosynthesis-related genes indicated similar transcriptional expression profiles in both cultivars. Our results revealed that phosphorylation modifications played critical roles in amylose and amylopectin biosynthesis, but the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.