Project description:Here is reported the first study of transcriptome analyses using the Illumina HiSeq 4000 platform for three kinds of wheat (G represents Strong gluten wheat, Z represents middle gluten wheat,R represents weak gluten wheat). The variation of wheat varieties with different gluten content is mainly shown in the content of gluten, flour is divided into high gluten powder ( > 30%), medium gluten powder (26%-30%) and low gluten powder ( < 20%), according to the wet gluten content. In total, over 102.6 Gb clean reads were produced and 114, 621 unigenes were assembled; more than 59,085 unigenes had at least one significant match to an existing gene model. Differentially expressed gene analysis identified 2339 and 2600 unigenes which were expressed higher or lower among strong gluten, middle gluten and weak gluten wheat. After functional annotation and classification, three dominant pathways including protein isomerase, antioxidase activity and energy metabolism, and 410 unigenes related to gluten strength polymerization of wheat were discovered. In strong-gluten wheat, low molecular weight subunit content is higher than weak-gluten wheat, and the activity of cysteine synthase and isomerase is increased, which may promote the cross-linking of low molecular weight protein to high molecular weight protein. Meanwhile, POD enzyme strengthens gluten network and CAT enzyme affects gluten polymerization, along with higher ATPase activity, which will provides energy for protein polymerization reaction in comparison of strong-gluten wheat and weak-gluten wheat. The accuracy of these RNA-seq data was validated by qRT-PCR analysis. These data will extend our knowledge of quality characteristics of wheat and provide a theoretical foundation for molecular mechanism research of wheat.
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:Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reductions of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are independently regulated to the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling.
Project description:Within the complex wheat flour proteome, the gluten proteins have attracted most of the attention because of their importance in determining the functional properties of wheat flour doughs and their roles in human health conditions such as celiac disease and food allergies. However, certain non-gluten proteins also trigger immunological responses but may be present in flour in low amounts or obscured by the more abundant gluten proteins in two-dimensional gels of total protein preparations. Non-gluten proteins were preferentially extracted from the flour with a dilute salt solution and separated by two-dimensional gel electrophoresis. Proteins in 172 gel spots were identified by tandem mass spectrometry after cleavage with trypsin or chymotrypsin. Fifty-seven different types of non-gluten proteins were identified, including 14 types that are known or suspected immunogenic proteins. The predominant proteins in 18 gel spots were gluten proteins. Transgenic wheat lines in which specific groups of gluten proteins were suppressed by RNA interference were used to estimate the amount of carry-over of gluten proteins in the salt-soluble protein fraction. Analysis of salt-soluble proteins from a transgenic line missing omega-1,2 gliadins demonstrated that certain omega-1,2 gliadins were present in large amounts in the salt-soluble fraction and obscured relatively small amounts of beta-amylase and protein disulfide isomerase. In comparison, analysis of a transgenic line in which alpha gliadins were absent revealed that glyceraldehyde-3 phosphate dehydrogenase was a moderately abundant protein that co-migrated with several alpha gliadins. The proteomic map of the non-gluten protein fraction of wheat flour developed in this study complements a proteomic map of the total flour proteins developed previously for the same cultivar. Knowing the identities of low abundance proteins in the flour as well as proteins that are hidden by some of the major gluten proteins on two-dimensional gels is critical for studies aimed at assessing the immunogenic potential of wheat flour and determining how the growth conditions of the plants affect the levels of specific immunogenic proteins in the flour.
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:Drought is among the most limiting factors for sustainable agricultural production. Water shortage at the onset of flowering severely affects the quality and quantity of grain yield of bread wheat (Triticum aestivum). Herein, we measured oxidative stress and photosynthesis-related parameters upon applying transient drought on contrasting wheat cultivars at the flowering initiation stage of ontogenesis. The sensitive cultivar showed ineffective water management and a more severe decline of photosynthesis. Apparently, the tolerant genotype used photorespiration to dissipate excessive light energy. The tolerant cultivar sooner induced superoxide dismutase and showed less inhibited photosynthesis. Such protective effect resulted in less affected yield and spectrum of seed proteome. The tolerant cultivar had a more stable gluten profile, which defines bread-making quality, upon drought. Drought caused the accumulation of medically relevant proteins: (i) components of gluten in the sensitive cultivar and (ii) metabolic proteins in the tolerant cultivar. We propose specific proteins as markers of drought tolerance for guiding efficient breeding: thaumatin-like protein, 14-3-3 protein, peroxiredoxins, peroxidase, FBD domain protein, and Ap2/ERF plus B3 domain protein.
Project description:Allohexaploid bread wheat (Triticum aestivum, L.) provides ~ 20% of calories consumed by humans. Hitherto lack of genome sequence for the three homoelogous and highly similar bread wheat genomes (A, B and, D) impeded expression analysis of the grain transcriptome. We used novel genome information to analyze the cell type specific expression of homeologous genes in the developing wheat grain.
Project description:Bread wheat (Triticum aestivum L.) is one of the most valuable cereal crops for the human consumption. Its grain storage proteins greatly impact bread quality, though may cause food intolerances or allergies in susceptible individuals. Consequently, the investigation of a proteome polymorphism among wheat varieties is important to spot the genotypes, which would be promising donors for the breeding of hypoallergenic cereals. Herein, we discovered diversity of grain proteins in three Ukrainian wheat cultivars: ‘Sotnytsia’, ‘Panna’ (both modern selection) and ‘Ukrainka’ (old landrace). Firstly, proteins were isolated with a SDS-containing buffer that allowed extraction of various groups of storage proteins (glutenins, gliadins, globulins and albumins). Secondly, the proteome was profiled by the two-dimensional gel electrophoresis, revealing 810 clearly-separated gel spots. Software-assisted analysis of gel images, showed 66 differentially abundant proteins. Using multi-enzymatic digestion, followed by the tandem mass spectrometry, we identified 49 differentially accumulated proteins. Parallel ultrahigh-performance liquid chromatography profiling and direct mass spectrometry quantification complemented the results. With this approach we quantified 127 proteins, 12 being differentially abundant. Principal component analysis confirmed genotype as a major source of variation in both cases. Non-gluten fraction was the most diverse among investigated bread wheat cultivars. Information from public databases of clinically relevant plant proteins highlighted variable groups of wheat allergens/toxins. Data suggested that one of the modern cultivars contained less health affecting proteins in grain. Finally, we proposed set of genetic landmarks for the development of DNA marker system, which will enable fast and efficient assessment of medical safety of multiple wheat genotypes to facilitate breeding programs.