A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a geater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when mem (8,20). SDS givesgood solubilization ofmembrane proteins (1), but because of its anionic nature, proteins solubilized in SDS cannot be applied directly to isoelectric focusing gels. In this paper, we examine four methods of membrane solubilization, two of which include SDS, for preparation of samples for isoelectric focusing. Solubilization ofa plasma membrane-enriched fraction from barley roots was used to assess the electrophoretic separations ofthese protein preparations on silver stained 2D gels.MATERIALS AND METHODS Plant Material. Seeds of barley (Hordeum vulgare L. cv California Mariout 72) were sown on moist cheesecloth above aerated Johnson's medium (10) and grown at 22°C in the dark for 7 d.Membrane Preparation. Microsomal suspensions were obtained from barley roots as previously described and the plasma membrane-enriched fraction was isolated based on the distribution of microsomal membranes on linear sucrose gradients (9). Microsomal suspensions were applied to discontinuous sucrose gradients consisting of 10 ml of 22% (w/w), 13 ml of 30% (w/ w), and 13 ml of 40% (w/w) sucrose in 1 mM DTT and 1 mM EDTA adjusted to pH 7.2 with Tris. The gradients were centrifuged for 2 h at 80,000g and the plasma membrane-enriched fraction was collected with a syringe by puncturing the centrifuge tube at the 30/40% sucrose interface. The fraction was washed with 150 mM KCI and pelleted as previously described (9).Solubilization of Membrane Proteins. The centrifuge tubes containing membrane pellets were inverted on ice for 10 min and excess supernatant removed before addition ofsolubilization buffer. Four methods were used to solubilize the plasma membrane-enriched fraction; all solutions containing ampholytes had 1.6% pH range 5 to 7 and 0.4% pH range 3.5 to 10.

BackgroundWheat 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.ResultsGrain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%.ConclusionsThis is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome the difficulties of matching peptides to gene sequences for members of highly similar, rapidly evolving storage protein families. Prospects for simplifying this process for routine analyses are discussed. The ability to measure expression levels for individual flour protein genes complements information gained from efforts to sequence the wheat genome and is essential for studies of effects of environment on gene expression.

Mitochondria contain thioredoxin (Trx), a regulatory disulfide protein, and an associated flavoenzyme, NADP͞Trx 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. Application of the mitochondrial extracts to mutant Trx affinity columns in conjunction with proteomics led to the identification of 50 potential Trx-linked proteins functional in 12 processes: photorespiration, citric acid cycle and associated reactions, lipid metabolism, electron transport, ATP synthesis͞ transformation, membrane transport, translation, protein assembly͞ folding, nitrogen metabolism, sulfur metabolism, hormone synthesis, and stress-related reactions. Almost all of these targets were also identified by a fluorescent gel electrophoresis procedure in which reduction by Trx can be observed directly. In some cases, the processes targeted by Trx depended on the source of the mitochondria. The results support the view that Trx acts as a sensor and enables mitochondria to adjust key reactions in accord with prevailing redox state. These and earlier findings further suggest that, by sensing redox in chloroplasts and mitochondria, Trx enables the two organelles of photosynthetic tissues to communicate by means of a network of transportable metabolites such as dihydroxyacetone phosphate, malate, and glycolate. In this way, light absorbed and processed by means of chlorophyll can be perceived and function in regulating fundamental mitochondrial processes akin to its mode of action in chloroplasts.T hioredoxins (Trxs) are small, widely distributed proteins with a redox-active disulfide group. Two types of Trx systems have been described in plants based on the source of reducing power: the ferredoxin͞Trx system located in chloroplasts [in which electrons from ferredoxin reduce Trx by means of the iron-sulfur enzyme ferredoxin͞Trx reductase (1-4)] and the extraplastidic NADP͞Trx system [whereby NADPH reduces Trx by means of the flavoenzyme NADP͞Trx reductase (NTR) (3, 4)].Trx has two main functions by nature of its ability to reduce specific S-S groups: (i) as a substrate for enzymes that catalyze reactions such as the reduction of ribonucleotides, methionine sulfoxide, and hydrogen peroxide; and (ii) as a regulator that alters the activity or other functional properties of interacting target proteins (1-4). The regulatory role of Trx is prominent in plants where the first redox-controlled enzyme was identified in chloroplasts Ͼ25 years ago (1, 2). Since that time, the number of Trx-regulated enzymes has steadily increased (2, 4). Recently developed proteomics-based approaches have accelerated progress in making possible the systematic identification of Trx-linked proteins in complex extracts. With the addition of previously u...

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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. We found that half of the identified proteins are involved in metabolism (20%), energy generation (17%), or cell structure (12%); these percentages are similar to those determined for the pollen transcriptome and this similarity is consistent with the idea that in addition to the mRNAs, the mature pollen grain contains proteins necessary for germination and rapid pollen tube growth. We identified ten proteins of unknown function, three of which are flower- or pollen-specific, and we identified nine proteins whose RNAs were absent from the transcriptome, seven of which are involved in metabolism, energy generation, or cell wall structure. Our work complements and extends recent analyses of the pollen transcriptome.

The origin of protein bodies in maize (Zen mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organefes or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endo-plasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or-insoluble proteins showed identical 114Clleucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough eadoplasmic reticulum. Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrfuation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosones were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained. During development of cereal seeds, storage proteins are synthesized in the endosperm and deposited in protein bodies (5, 9, 19). Based on electron micrographs of developing wheat endo-sperm, Morton and co-workers (20) concluded that these protein bodies originated in specialized protein-forming organelles (plas-tids) which contained polyribosomes distinct from those of the general ER. An ultrastructural study of developing maize seeds led Khoo and Wolf (11) to conclude that the endosperm protein bodies originated in vesicles produced by RER or formed at the enlarged ends ofRER cisterna. Recent experiments have demonstrated that protein bodies in maize (3, 13) and barley endosperm (2, 6) contain the major prolamine proteins and that these proteins are synthesized by membrane-bound polyribosomes. In some of these studies membrane bound polyribosomes were isolated from the particulate fraction that pelleted between 500 and 37,000g2 (2, 6, 13). In another study polyribosomes were isolated from a protein body fraction obtained by continuous sucrose gradient centrifugation (3). Because of the attachment of ribosomes to the membrane surrounding protein bodies, several investigators concluded that 'Journal Paper No. 7052 of the Purdue University Experiment Station. 2 Abbreviation: g gravitational force determined at Ray. protein bodies are highly differentiated sites of storage protein synthesis (3, 20, 27), and furthermore that mRNAs isolated from "protein body" polyribosomes represent a homogene...

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