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Background: Flour quality is largely determined by the gluten proteins, a complex mixture of proteins consisting of high molecular weight-glutenin subunits (HMW-GS), low molecular weight-glutenin subunits (LMW-GS), and a-, ?-, and ?-gliadins. Detailed proteomic analyses of the effects of fertilizer and high temperature on individual gliadin and glutenin protein levels are needed to determine how these environmental factors influence flour quality. Results: Wheat plants (Triticum aestivum L. cv. Butte 86) were grown in greenhouses under moderate and high temperature regimens with and without post-anthesis fertilizer. Quantitative two-dimensional gel electrophoresis was used to construct accumulation profiles in developing endosperm for the entire complement of gluten proteins identified previously by tandem mass spectrometry. Amounts of individual gliadins and glutenins were also determined in flour produced under each of the regimens. Under all environmental regimens, most HMW-GS, LMW-GS, ?- and ?-gliadins accumulated rapidly during early stages of grain development and leveled off during middle stages of development. A subset of LMW-GS showed a second distinct profile, accumulating throughout development, while a-gliadins showed a variety of accumulation profiles. In flour, fourteen distinct gluten proteins responded similarly to fertilizer, high temperature, and high temperature plus fertilizer. The majority of HMW-GS and ?-gliadins and some a-gliadins increased while two LMW-GS and a minor ?-gliadin decreased. Fertilizer did not influence gluten protein accumulation under high temperature conditions. Additionally, the effects of fertilizer and high temperature were not additive; very few changes were observed when plants that received fertilizer were subjected to high temperature. Conclusions: Although post-anthesis temperature and fertilizer have very different effects on grain development and yield, the two treatments elicit surprisingly similar effects on the accumulation of gluten proteins. The similarity of the responses to the different treatments is likely due to source-sink activities of nitrogen reserves in the wheat plant. Because each protein that showed a response in this study is linked to a gene sequence, the work sets the stage for transgenic studies that will better elucidate the roles of specific proteins in flour quality and in the response to the environment. [ABSTRACT FROM AUTHOR]

Wheat protein is a technologically challenging substrate for food and nonfood applications because of its compositional diversity and susceptibility to denaturation. Genetic modification could be used to create cultivars capable of producing more uniform or focused and novel protein compositions targeted to nonfood uses. These lines could serve as expression systems for specific high‐molecular‐weight (HMW) protein polymers and would be new crops leading to more diverse agricultural opportunities. However, fundamental changes to the molecular architecture in such wheat seeds could also result in separation and processing issues, such that conventional methods of protein enrichment may need modification or even reinvention. Enriched gluten protein fractions were prepared from Bobwhite lines modified to overproduce HMW glutenin subunits Dx5 and/or Dy10. These lines serve as experimental models to test various approaches that may be taken for protein polymer enrichment. However, conventional wheat gluten enrichment based on the glutomatic as a small model of industrial methods was incapable of producing enrichment for any of the tested meal or flour, including that from the non‐transformed parent Bobwhite. Mixing in the mixograph or farinograph failed to produce standard patterns for whole kernel meal and straight‐run flour, and the normal cohesiveness of dough expected from these devices was not observed. Microscopy of stained dough samples revealed severely limited formation of normal protein networks, a capability crucial to conventional separation technology. Particle size analysis of whole kernel meal revealed a higher resistance to milling for the altered lines. Higher drying rates, lower farinograph moisture absorption, and increased thermal transition temperatures were observed. These data suggested that the native architecture of these new forms was more tightly constructed with reduced capacity for alteration by hydration and input of mechanical energy. An alternative enrichment method featuring solvation in SDS and precipitation in acetone produced coagulated (Bobwhite) or partially coagulated protein (transgenic lines producing Dx5 or Dy10) enriched to 78–85% protein with high yield. [ABSTRACT FROM AUTHOR]

Abstract: Total protein extracts of wheat endosperm are widely used for the analysis of the highly abundant gliadins and glutenins. In this review, the most popular total endosperm extraction methods are compared for their effectiveness in proteome coverage. A drawback of total endosperm extracts is that the enormous dynamic range of protein abundance limits the detection, quantification, and identification of low abundance proteins. Protein fractionation is invaluable for improving proteome coverage, because it reduces sample complexity while enriching for specific classes of less abundant proteins. A wide array of techniques is available for isolating protein subpopulations. Sequential extraction is a method particularly suited for subfractionation of wheat endosperm proteins, because it takes advantage of the specific solubility properties of the different classes of endosperm proteins. This method effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. This approach is suitable for defining the biochemical processes associated with amyloplasts, specialized organelles in the endosperm that function in the synthesis and storage of starch. Subproteome fractionation, when combined with 2-DE and protein identification, provides a powerful approach for defining endosperm protein composition and providing new insights into cellular functions. [Copyright &y& Elsevier]

A KCl-soluble, albumin/globulin fraction of wheat (Triticum aestivum L.) starchy endosperm was further separated into a methanol-insoluble fraction that contained metabolic proteins and a methanol-soluble fraction that contained “chloroform-methanol” or CM-like proteins. Reduction of the disulfide bonds of the CM proteins with thioredoxin or dithiothreitol altered their properties so that, like the metabolic proteins, they were insoluble in methanol. Glutathione had little effect, indicating dithiol specificity. Proteomic analysis of the CM protein fraction revealed the presence of isoforms of low molecular weight disulfide proteins (α-amylase, α-amylase/trypsin and WCI proteinase inhibitors, lipid transfer proteins, γ-thionins), stress enzymes (Cu-Zn superoxide dismutase and peroxidase), storage proteins (α-, γ- and ω-gliadins, low molecular weight glutenin subunits and globulins of the avenin N9 type), and a component of protein degradation (polyubiquitin). These findings support the view that, in addition to modifying activity and increasing protease sensitivity, reduction by thioredoxin alters protein solubility, thereby promoting processes of the grain starchy endosperm, notably the mobilization of reserves during germination and seedling development. [ABSTRACT FROM PUBLISHER]

Application of a thiol-specific probe, monobromobimane, with proteomics and enzyme assays led to the identification of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identified targets, 12 not known to be thioredoxin-linked, function in a spectrum of processes: metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed. [Copyright &y& Elsevier]

Biosynthesis and accumulation of seed storage proteins such as the wheat glutens depend on the activity of a variety of other proteins, including chaperones and foldases. cDNA probes and antibodies to two chaperone proteins and a foldase were used to follow mRNA and protein accumulation in developing grains of wheat (Triticum aestivum, cvs Cheyenne and Butte). Endosperm was separated from other grain components and protein accumulation was analyzed on a per mg fresh weight basis. The ER resident chaperone BiP (binding protein) and foldase PDI (protein disulfide isomerase) accumulated to maximal levels in the middle stage of endosperm development, a period of rapid cell expansion and storage protein accumulation, whereas levels of a cytosolic chaperone, HSP70, remained relatively constant throughout grain development. In contrast, nucleoside diphosphate kinase (NDK), a cytosolic enzyme needed for synthesis of nucleoside triphosphates, accumulated early in endosperm development during the period of nuclear division and cell formation. When analyzed as a fraction of total protein the relative abundance of all four proteins peaked early in grain development and then declined. Accumulation of mRNA for the four proteins also peaked early in grain development. Although BiP and PDI formed a declining percentage of total protein as storage protein accumulated, their pattern of accumulation was compatible with a proposed role as catalysts for storage protein folding and accumulation in the ER. [ABSTRACT FROM AUTHOR]

The effects of high temperature on accumulation of the 70-kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17-kDa NDK were co-purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants (Triticum aestivum, cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid-grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1-day gradual increase to 40°C. The effects of a 5-day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post-anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue. [ABSTRACT FROM AUTHOR]

Abstract: The accumulation of KCl-soluble/methanol-insoluble albumins and globulins was investigated in the endosperm of developing wheat (Triticum aestivum, L. cv. Butte 86) grain produced under a moderate (24°C/17°C, day/night) or a high temperature regimen (37°C/28°C) imposed from 10 or 20 days post-anthesis (dpa) until maturity. Proteins were separated by 2-DE and developmental profiles for nearly 200 proteins were analyzed by hierarchical clustering. Comparison of protein profiles across physiologically equivalent stages of grain fill revealed that high temperature shortened, but did not substantially alter, the developmental program. Accumulation of proteins shifted from those active in biosynthesis and metabolism to those with roles in storage and protection against biotic and abiotic stresses. Few proteins responded transiently when plants were transferred to the high temperature regimens, but levels of a number of proteins were altered during late stages of grain development. Specific protein responses depended on whether the high temperature regimens were initiated early or mid development. Some of the heat responsive proteins have been implicated in gas bubble stabilization in bread dough and others are suspected food allergens. [Copyright &y& Elsevier]

The role of thioredoxin in wheat starchy endosperm was investigated utilizing two proteomic approaches. Thioredoxin targets were isolated from total KCl-soluble extracts of endosperm and flour and separated by 2-DE following (1) reduction of the extract by the NADP/thioredoxin system and labeling the newly generated sulfhydryl (SH) groups with monobromobimane (mBBr), and, in parallel, (2) trapping covalently interacting proteins on an affinity column prepared with mutant thioredoxin h in which one of the active site cysteines was replaced by serine. The two procedures were complementary: of the total targets, one-third were observed with both procedures and one-third were unique to each. Altogether 68 potential targets were identified; almost all containing conserved cysteines. In addition to confirming known interacting proteins, we identified 40 potential thioredoxin targets not previously described in seeds. A comparison of the results obtained with young endosperm (isolated 10 days after flowering) to those with mature endosperm (isolated 36 days after flowering) revealed a unique set of proteins functional in processes characteristic of each developmental stage. Flour contained 36 thioredoxin targets, most of which have been found in the isolated developing endosperm. [Copyright &y& Elsevier]

The effect of high temperature on starch accumulation, starch granule populations, and expression of genes encoding key enzymes for starch biosynthesis was examined during grain development in wheat (Triticum aestivum L. cv. Butte 86). High temperature applied from anthesis to maturity reduced the duration of starch accumulation. Starch accumulation ceased approximately 6 days earlier for grain produced under a 37/17 °C (day/night) regimen and 21 days earlier under a 37/28 °C (day/night) regimen than for grain produced under a 24/17 °C (day/night) regimen. Compared to the 24/17 °C regimen, starch content was approximately 19% less for mature grain produced under the 37/17 °C regimen and 58% less under the 37/28 °C regimen. Based on relative volume, the smaller type B starch granules were the predominant class in mature grain produced under the 24/17 and 37/17 °C regimens, whereas the larger type A granules were predominant in grain produced under the 37/28 °C regimen. Under the 24/17 °C regimen, steady state transcript levels for ADP-glucose pyrophosphorylase, starch synthases I, II, and III, granule-bound starch synthase, and starch branching enzymes I and II were highest from 12–16 days post-anthesis (dpa). Under the 37/17 °C regimen, steady state levels of these transcripts followed the same temporal pattern, but were substantially lower. Under the 37/28 °C regimen, transcript levels peaked earlier, at 7 dpa. The high temperature regimens reduced the relative levels of transcripts for starch synthase more than the other starch biosynthetic enzymes. [Copyright &y& Elsevier]