The need for agriculture phenotyping: "moving from genotype to phenotype".

Unlabelled: Increase in the world population has called for the increased demand for agricultural productivity. Traditional methods to augment crop and animal production are facing exacerbating pressures in keeping up with population growth. This challenge has in turn led to the transformational change in the use of biotechnology tools to meet increased productivity for both plant and animal systems.

Comparative proteomic analysis of the effect of temperature and fertilizer on gliadin and glutenin accumulation in the developing endosperm and flour from Triticum aestivum L. cv. Butte 86.

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 α-, γ-, and ω-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.

Differential effects of a post-anthesis fertilizer regimen on the wheat flour proteome determined by quantitative 2-DE.

Background: Mineral nutrition during wheat grain development has large effects on wheat flour protein content and composition, which in turn affect flour quality and immunogenic potential for a commodity of great economic value. However, it has been difficult to define the precise effects of mineral nutrition on protein composition because of the complexity of the wheat flour proteome. Recent improvements in the identification of flour proteins by tandem mass spectrometry (MS/MS) and the availability of a comprehensive proteome map of flour from the US wheat Butte 86 now make it possible to document changes in the proportions of individual flour proteins that result from the application of mineral nutrition.

High temperature during grain fill alters the morphology of protein and starch deposits in the starchy endosperm cells of developing wheat (Triticum aestivum L.) grain.

High temperature during grain fill reduces wheat yield and alters flour quality. Starchy endosperm cell morphology was investigated in wheat ( Triticum aestivum L. 'Butte 86') grain produced under a 24/17 or 37/28 °C day/night regimen imposed from anthesis to maturity to identify changes in cell structure related to the functional properties of flour.

Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry.

Background: Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences.

Finding the "bio" in biobased products: electrophoretic identification of wheat proteins in processed products.

Verification of the biocontent in biobased or "green" products identifies genuine products, exposes counterfeit copies, supports or refutes content claims, and ensures consumer confidence. When the biocontent includes protein, elemental nitrogen analysis is insufficient for verification since non-protein, but nitrogen-rich, content also may be present. However, the proteins can be extracted, separated by electrophoretic methods, and detected by UV absorption, protein stain, or immunoblotting.

Thioredoxin targets in plants: the first 30 years.

The turn of the century welcomed major developments in redox biology. In plants, proteomics made possible the identification of proteins linked to thioredoxin (Trx), initially in chloroplasts and then other cell compartments. Two procedures, one based on thiol specific probes and the other on mutant Trx proteins, facilitated the labeling or isolation of potential Trx targets that were later identified with proteomic approaches.

Surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients.

Wheat starch is used to make baked products for celiac patients in several European countries but is avoided in the United States because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye, and barley proteins and products that contain them. These proteins are capable of initiating damage to the absorptive lining of the small intestine in CD patients, apparently as a consequence of undesirable interactions with the innate and adaptive immune systems.

Thioredoxin-linked proteins are reduced during germination of Medicago truncatula seeds.

Germination of cereals is accompanied by extensive change in the redox state of seed proteins. Proteins present in oxidized form in dry seeds are converted to the reduced state following imbibition. Thioredoxin (Trx) appears to play a role in this transition in cereals.

Extraction of wheat endosperm proteins for proteome analysis.

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.

Thioredoxin target proteins in chloroplast thylakoid membranes.

In recent years, impressive progress has been made in the identification of thioredoxin-linked proteins. However, due to technical difficulties inherent in working with hydrophobic proteins, identifications so far have been restricted to proteins in the soluble fraction. Thus, our knowledge of redox regulated membrane proteins is quite limited.

Proteome of amyloplasts isolated from developing wheat endosperm presents evidence of broad metabolic capability.

By contrast to chloroplasts, our knowledge of amyloplasts--organelles that synthesize and store starch in heterotrophic plant tissues--is in a formative stage. While our understanding of what is considered their primary function, i.e.

A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts.

A growing number of processes throughout biology are regulated by redox via thiol-disulfide exchange. This mechanism is particularly widespread in plants, where almost 200 proteins have been linked to thioredoxin (Trx), a widely distributed small regulatory disulfide protein. The current study extends regulation by Trx to amyloplasts, organelles prevalent in heterotrophic plant tissues that, among other biosynthetic activities, catalyze the synthesis and storage of copious amounts of starch.

Proteome mapping of mature pollen of Arabidopsis thaliana.

The male gametophyte of Arabidopsis is a three-celled pollen grain that is thought to contain almost all the mRNAs needed for germination and rapid pollen tube growth. We generated a reference map of the Arabidopsis mature pollen proteome by using multiple protein extraction techniques followed by 2-DE and ESI-MS/MS. We identified 135 distinct proteins from a total of 179 protein spots.

Developmental changes in the metabolic protein profiles of wheat endosperm.

A combined two-dimensional gel electrophoresis-mass spectrometry approach was utilized to identify over 250 proteins of wheat (Triticum aestivum L., cv. Butte 86) starchy endosperm that participate in 13 biochemical processes: ATP interconversion reactions, carbohydrate metabolism, cell division, cytoskeleton, lipid metabolism, nitrogen metabolism, protein synthesis/assembly, protein turnover, signal transduction, protein storage, stress/defense, transcription/translation, and transport.

Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches.

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.

Thioredoxin reduction alters the solubility of proteins of wheat starchy endosperm: an early event in cereal germination.

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.

Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria.

Mitochondria contain thioredoxin (Trx), a regulatory disulfide protein, and an associated flavoenzyme, NADP/Trx reductase, which provide a link to NADPH in the organelle. Unlike animal and yeast counterparts, the function of Trx in plant mitochondria is largely unknown. Accordingly, we have applied recently devised proteomic approaches to identify soluble Trx-linked proteins in mitochondria isolated from photosynthetic (pea and spinach leaves) and heterotrophic (potato tubers) sources.

Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics.

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).

Identification of wheat endosperm proteins by MALDI mass spectrometry and LC-MS/MS.

The commercial value of the wheat crop is a function of the quality and amount of the storage protein and starch present in the grain, which in turn are influenced by environmental conditions during grain-fill. To understand how environment modifies the size and composition of wheat grains, we set out to identify the key metabolic and regulatory proteins in developing grain. We present results of initial studies aimed at establishing instrument conditions that will allow us to identify cytoplasmic proteins present in wheat endosperm.