Summary Two-dimensional electrophoresis (2-DE) combined with mass spectrometry was used to identify proteins related to the development of mealiness in peaches. Five proteins were identified that differed significantly in amount between peaches that had mealy flesh and those that remained juicy following 3 weeks of storage at 5°C and subsequent ripening for 3 – 4 d at 23°C. The accumulation of ACC oxidase (ACO), a key enzyme of ethylene biosynthesis, decreased in mealy peaches. Immunoblots probed with antibodies against ACO confirmed the 2-DE results, and showed that the relationship between ACO levels and mealiness also existed within individual fruit. Phosphoglycerate kinase, an enzyme in the glycolytic pathway, was reduced in amount in mealy fruit. In contrast, two heat shock proteins and hydroxymethylbutenyl-4-diphosphate synthase increased in amounts in mealy peaches. The results regarding ACO levels are of interest due to the confirmed association of mealiness and ethylene and, given the regulatory effect of ethylene on cell wall degrading enzymes, suggest that the reduction in ACO expression during cold storage and subsequent ripening may be pivotal in the development of mealiness.

A survey of EST databases identified 16 9-kDa non-specific lipid transfer proteins (nsLTPs) expressed in developing grain from the US spring wheat Butte 86. Two of the most abundant sequences encoded nsLTPs similar to proteins identified previously by two-dimensional gel electrophoresis/mass spectrometry (2-DE/MS). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to examine the expression of these nsLTPs in developing grain or endosperm produced under controlled temperature and fertilizer regimens. Under a moderate (24°C/17°C day/night) temperature regimen, transcripts accumulated late in grain development and achieved highest levels as grain reached maximum dry weight. When high temperature regimens (37°C/28°C day/night) were imposed from anthesis or 15 days post-anthesis (DPA) until maturity, maximum transcript levels were as much as 9-fold higher. Post-anthesis fertilizer had relatively small effects on transcript levels. Accumulation profiles for 9-kDa nsLTPs generated by 2-DE were consistent with transcript profiles. The nsLTPs increased 2 to 3.5-fold in amount in endosperm in response to high temperatures but showed little change in response to fertilizer. Flour from grain produced under the high temperature regimens also contained greater amounts of nsLTPs. Increases in the levels of nsLTPs in flour as a result of environmental conditions during grain fill may have important implications for flour quality and allergenic potential.

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. the biosynthesis and degradation of starch, has increased dramatically in recent years, relatively little is known about other biochemical processes taking place in these organelles. To help fill this gap, a proteomic analysis of amyloplasts isolated from the starchy endosperm of wheat seeds (10 d post-anthesis) has been conducted. The study has led to the identification of 289 proteins that function in a range of processes, including carbohydrate metabolism, cytoskeleton/plastid division, energetics, nitrogen and sulphur metabolism, nucleic acid-related reactions, synthesis of various building blocks, protein-related reactions, transport, signalling, stress, and a variety of other activities grouped under 'miscellaneous'. The function of 12% of the proteins was unknown. The results highlight the role of the amyloplast as a starch-storing organelle that fulfills a spectrum of biosynthetic needs of the parent tissue. When compared with a recent proteomic analysis of whole endosperm, the current study demonstrates the advantage of using isolated organelles in proteomic studies.

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. Using proteomics and immunological methods, we identified the components of the ferredoxin/Trx system (ferredoxin, ferredoxin-Trx reductase, and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin-NADP reductase. However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxin-Trx reductase and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the identification of 42 potential Trx target proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids, and nucleotides; protein folding; and several miscellaneous reactions. The results suggest a mechanism whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves.

Hard red spring wheat (Triticum aestivum cv Butte86) was grown under controlled environmental conditions and grain produced under 24/17°C, 37/17°C or 37/28°C day/night regimens with or without post-anthesis N supplied as NPK. Flour proteins were analyzed and quantified by differential fractionation and RP-HPLC, and endosperm proteins were assessed by two-dimensional gel electrophoresis (2-DE). High temperature or NPK during grain fill increased protein percentage and altered the proportions of S-rich and S-poor proteins. Addition of NPK increased protein accumulation per grain under the 24/17°C but not the 37/28°C regimen. However, flour protein composition was similar for grain produced with NPK at 24/17°C or 37/28°C. 2-DE of gluten proteins during grain development revealed that NPK or high temperature increased the accumulation rate for S-poor proteins more than for S-rich proteins. Flour S content did not indicate S-deficiency, however, and addition of post-anthesis S had no effect on protein composition. Although, high-protein flour from grain produced under the 37/28°C regimen with or without NPK had loaf volumes comparable to flour produced at 24/17°C with NPK, mixing tolerance was decreased by the high temperature regimen.

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.

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. Endosperm protein populations were compared at early (10 days post-anthesis, dpa) and late (36 dpa) stages of grain development. Analysis of protein number and spot volume revealed that carbohydrate metabolism, transcription/translation, and protein synthesis/assembly were the principal endosperm functions at 10 dpa followed by nitrogen metabolism, protein turnover, cytoskeleton, cell division, signal transduction, and lipid metabolism. Carbohydrate metabolism and protein synthesis/assembly were also major functions at 36 dpa, but stress/defense and storage were predominant. The results provide insight into biochemical events taking place during wheat grain development and highlight the value of proteomics in characterizing complex biochemical processes. Further, the proteome maps will facilitate future studies addressing the effects of genetic and environmental factors on the development and quality of wheat grain.

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.

To understand wheat dough protein behavior under dual mixing and thermal treatment, solubility of Mixolab-dough proteins were investigated using nine extraction buffers of different dissociation capacities. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) demonstrated that overall changes of protein fractions and dynamic responses of specific proteins during dough processing were well reflected by their solubility variations. After starch pasting, the abundance of 0.5 M NaCl extractable proteins were decreased except for six protein groups including α-amylase inhibitors and superoxide dismutase (SOD). The solubility loss of glutenin proteins at C3 (32 min; 80 ℃) was mainly ascribed to the un-extractable HMW-GSs, LMW-GSs, globulin and triticin, while the extract yield of α-, β-, γ-gliadins and avenin-like proteins (ALPs) increased after starch pasting. Differential responses of dough proteins to extraction systems provides the basis for further exploring wheat protein dynamics in processing.

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 (alpha-amylase, alpha-amylase/trypsin and WCI proteinase inhibitors, lipid transfer proteins, gamma-thionins), stress enzymes (Cu-Zn superoxide dismutase and peroxidase), storage proteins (alpha-, gamma- and omega-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.

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. 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 synthesis/transformation, membrane transport, translation, protein assembly/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.

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.

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.

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.

Germin gene expression in barley (Hordeum vulgare L.) seedlings responds to developmental and environmental cues. During seed germination, germin mRNA levels were maximal 2 d after the start of imbibition in control seedlings and declined to low levels by 6 d. When seeds were sown in the presence of 200 mM NaCl, germin mRNA levels were also maximal after 2 d, but NaCl treatment, which slowed seedling growth, prolonged germin gene expression for an additional 1 d. In 4-d-old seedlings, germin mRNA levels were highest in roots and higher in the vascular transition region than in shoots. In roots of 6-d-old seedlings, germin gene expression was regulated by salt shock and plant growth regulators. Induced germin mRNA levels were maximal 8 h after treatment with NaCl, salicylate, methyl salicylate, or methyl jasmonate and 4 h after treatment with abscisic acid and indoleacetic acid. Like germin mRNA, dehydrin mRNA levels were maximal 8 h after NaCl treatment. In contrast, peroxidase mRNA levels declined to less than control levels within 30 min of treatment. Germin gene expression is regulated developmentally by salt stress and by treatments with plant hormones. Since germin is an oxalate oxidase, these result imply that oxalate has important roles in plant development and homeostasis.

Germin gene expression is induced in wheat (Triticum aestivum L.) leaves by powdery mildew (Erysiphe graminis f. sp. tritici) infection. Germin is a protein marker for early cereal development and is an oxalate oxidase, an enzyme that catalyzes the conversion of oxalate to CO2 and H2O2. The induction of germin gene expression by powdery mildew infection is consistent with the importance of H2O2 to plant defense and identifies a mechanism for the elevation of H2O2 levels in wheat leaves. Germin mRNA levels increased 2 d after inoculation of seedlings with powdery mildew and continued to increase throughout an 8-d time course. The increase in accumulation of germin mRNA was accompanied by an increase in the germin oligomer, which reached maximal levels by d 6. An increase in oxalate oxidase activity paralleled germin oligomer accumulation. Germin gene expression was induced in a relatively resistant cultivar (Bobwhite) as well as in a susceptible cultivar (Cheyenne), suggesting that the induction of germin gene expression is an indicator of powdery mildew infection rather than cultivar resistance.

Salt stress alters the synthesis (in vivo labeling with [35S]Met of a large number of polypeptides in roots of CM72, a relatively salt-tolerant barley (Hordeum vulgare L.) cultivar (Hurkman and Tanaka, 1987; Hurkman et al., 1989). The most significant changes are related to the biosynthesis of three polypeptides related to germin, an approximately 130kD homopentameric protein comprised of subunits of about 26 kD (McCubbin et al., 1987) that arises in the embryos of wheat seeds during germination (Grzelczak and Lane, 1984). Two 26-kD (pI 6.3 and 6.5) polypeptides accumulate and one 27-kD (pI 5.6) polypeptide declines (quantitatively in stained gels) during salt stress (Hurkman et al., 1991). NH2-terminal amino acid sequences in the 26-kD polypeptides share strong residue identity with NH2-terminal amino acid sequences in wheat germin (Hurkman et al., 1991). In addition, antisera against the barley 26-kD polypeptides react with wheat germin 26-kD polypeptides and antiserum against wheat germin reacts with the 27-kD barley polypeptide (Hurkman et al., 1991). Like wheat germin (aikaran et al., 1990), the barley 26-kD polypeptides are glycosylated (Hurkman et al., 1991), and like wheat germin oligomers (Grzelczak and Lane, 1984), the putative barley germin oligomers are protease resistant (Hurkman et al., 1988). A cDNA library constructed in Xgtl 1 using poly(A)+ RNA isolated from roots of 6-d-old CM72 seedlings grown in the presence of NaCl (Table I) was screened with a cDNA that encodes wheat germin gf-2.8 mRNA (Lane et al., 1991). A single, full-length cDNA encoding a barley germin subunit was obtained. The sequence for this barley germin cDNA is 98.5% similar to that of wheat germin gf-2.8, and it likely encodes one of the 26or 27-kD polypeptides whose levels change during salt stress. The wheat and barley germins share homology with the spherulins, putative cell-wall proteins in the slime mold Physarum polycephalum that increase during spherulation, a process brought on by environmental stresses, including osmotic stress (Lane et al., 1991). A rootspecific transcript encoding an mRNA that decreases during