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A biochemical study of the main durum wheat milling fractions (bran, embryo, and semolina) showed that peroxidases (POD) were present in multiple forms in the kernel and appeared to be tissue specific: one form for the embryo, one for the endosperm, one for the subaleuronic layer, and one for the outer layers. Large varietal differences were found regarding both the composition and the POD activity. POD activity, detected by diaminobenzidine, was found mainly in the cell wall of the subaleurone layer and inside some specific, differentiated cells of the embryo. Immuno-localization with antibodies of durum wheat POD showed the presence of POD in several layers of the pericarp (epidermis) and the seed coat (testa), in the embryo, and also in the endosperm. In this latter tissue, the staining intensity decreased gradually from the outer layers toward the center of the kernel. The localization of POD in durum wheat kernel suggests specific functions for different forms.

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S-S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the alpha and beta fractions were most potent, followed by the gamma and omega types. Thioredoxin mitigated the allergenicity associated with the major protein fractions-i.e, the gliadins (including the alpha, beta, and gamma types) and the glutenins-but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP-thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously-i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

Protease inhibitors, including the Kunitz and Bowman-Birk trypsin inhibitorsof soybean, can be reduced by the NADP/thioredoxin system (NADPH, thioredoxin, and NADP-thioredoxin reductase) from either Escherichia coli or wheat germ. In the present study, we report that, when reduced by thioredoxin, the Kunitz and Bowman-Birk soybean trypsin inhibitors lose their ability to inhibit prytsin. Moreover, the reduced form of the inhibitors showed increased susceptibility to heat and proteolysis by either subtilisin or a protease preparation from germinating wheat seeds

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*INRA Unité de Biochimie et Biologie Moléculaire des Céréales Montpellier (FRA) Diffusion du document : INRA Unité de Biochimie et Biologie Moléculaire des Céréales Montpellier (FRA); International audience

Thioredoxin of the h-type — earlier linked to the reduction of wheat (Triticum durum Desf. cv. Monroe) endosperm proteins — was converted from an oxidized to a partially reduced state during germination and seedling development. While the abundance of thioredoxin progressively decreased during this period, the availability of reducing equivalents, defined as the product of the relative abundance of thioredoxin and the percent reduction, increased. The amount of the enzyme catalyzing the reduction of thioredoxin h (NADP-thioredoxin reductase) remained constant. The activities of enzymes generating the NADPH needed for the reduction of thioredoxin (glucose 6-phosphate and 6-phosphogluconate dehydrogenases) increased. The level of thioredoxin h in the endosperm appeared to be controlled by the embryo via hormones. Gibberellic acid enhanced the disappearance of thioredoxin, whereas abscisic acid showed the opposite effect. Moreover, uniconazole, an inhibitor of gibberellic acid synthesis, slowed seedling growth and inhibited the disappearance of thioredoxin in a manner reversible by gibberellic acid. The results are consistent with a role for thioredoxin h in initiating the mobilization of nitrogen and carbon needed for germination and seedling development.

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

Publisher Summary This chapter focuses on the thioredoxin and seed proteins. Storage proteins, which are formed and stored within seeds, serve as the main source of nitrogen and as a primary source of carbon for the germination and growth of seedlings. Storage proteins are synthesized after pollination and accumulate during seed maturation. The proteins are mobilized, proteolytically degraded, and utilized when conditions are favorable for germination. In the case of cereals, the bulk of the storage proteins are water insoluble after the grains mature. In most other types of seeds, the proteins remain soluble and enclosed within a membrane, thereby creating a structure known as a protein body. In some cereals, the protein body membrane is disrupted during maturation and drying of the grain, thereby exposing the storage proteins to endogenous proteases of the endosperm. Disulfide bonds are one of the main stabilizing forces in storage proteins. The formation of disulfide bonds appears to function in cereals to provide increased structural stability on the one hand and decreased solubility on the other. Both features provide protection against proteolysis.

Lipoic acid was found to be present at low levels in wheat germ (ca. 0.1 ppm) but was not detected in flour or semolina. The reduced form of lipoic acid provided the reducing equivalents for thioredoxin in the reduction of proteins of wheat endosperm (albumins, globulins, gliadins, and glutenins). The results indicate that, while lipoic acid could function to reduce thioredoxin physiologically, its importance to technologies based on flour or semolina is questionable

The 2S albumin from the endosperm of castor seed (Ricinus communis L.) seed was reduced by thioredoxin from either wheat germ or Escherichia coli. The 2S protein is made up of a large (approx. 7 kDa) subunit that contains two intramolecular disulfides and a small (approx. 4 kDa) subunit that lacks intramolecular disulfides. The two subunits are joined by at least one intermolecular disulfide bond. Thioredoxin could be reduced either enzymically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol. Reduced glutathione and glutaredoxin (from E. coli) were without effect. The ability of the 2S protein to undergo reduction by thioredoxin was demonstrated by a direct reduction procedure based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by an enzymatic procedure in which reduction is linked to activation of chloroplast NADP-malate dehydrogenase. Analyses indicated that thioredoxin actively reduced the intramolecular disulfides of the 2S large subunit, but was ineffective in reducing the intermolecular disulfide(s) that connect the large to the small subunit. These findings extend the role of thioredoxin to the reduction of a seed protein that is widely distributed in oil producing plants.

Gliadins and glutenins, the major storage proteins of wheat endosperm (Triticum durum, Desf. cv Monroe), were reduced in vitro by the NADP/thioredoxin system (NADPH, NADP-thioredoxin reductase and thioredoxin; in plants, the h type) from either the same source or the bacterium Escherichia coli. A more limited reduction of certain members of these protein groups was achieved with the reduced form of glutathione or glutaredoxin, a protein known to replace thioredoxin in certain bacterial and mammalian enzyme systems but not known to occur in higher plants. Endosperm extracts contained the enzymes necessary to reduce NADP by the oxidative pentose phosphate pathway (hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase). The gliadins and glutenins were also reduced in vivo during germination--an event that accompanied their proteolytic breakdown. The results suggest that thioredoxin, reduced by NADPH generated via the oxidative pentose phosphate pathway, functions as a signal in germination to enhance metabolic processes such as the mobilization of storage proteins and, as found earlier, the activation of enzymes.

Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5‘,5‘-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.

Gas chromatographic and mass spectrometric analyses showed that two low molecular weight proteins, extracted from glutenins of two durum wheat cultivars, contained strongly bound lipids with particular composition in comparison to semolina lipids. The percentages of palmitic acid stearic acid, and oleic acid were high; Various fatty alcohols and hydrocarbons were also present in the purified proteins; the amount of dodecanol, especially in one sample, was very high

Polyacrylamide-slab gel electrophoresis showed that the nature of peroxidases in wheat is a genetic character; different genomes control the synthesis of specific peroxidases. The growing conditions have no influence on isozyme patterns. Within a single species the electrophoretic patterns of peroxidases may differ from one variety to another. Thus, two classes of Triticum aestivum can be distinguished. With Triticum durum the dissimilarity of enzymograms is particularly important.

Semolina samples were obtained from several durum wheat cultivars. The cooking quality of the spaghetti prepared from the semolina showed both inter and intravarietal differences. Proteins from semolina were extracted sequentially and both the reactive and total SH plus SS content of the protein extracts determined amperometrically. The albumin-globulin fractions contained the highest amounts of both reactive and total SH plus SS groups. The total SH plus SS content was higher in the gliadins than in the glutenins but the latter contained higher amounts of reactive SH plus SS groups. A highly significant correlation was found between the total amount of SH plus SS content of the glutenins and the cooking quality of the samples (r=0.835): This might be a functional relation.

Durum wheat glutenin fractions, composed of two low molecular weight proteins DSG-1 and DSG-2 (durum wheat (Triticum durum Desf) sulphurrich glutenin fractions) were extracted from semolina samples using a low concentration of Na tetradecanoate after extracting albumins, globulins and gliadins. DSG proteins have a highSH plus SS content. A highly significant correlation was found between the SH plus SS content of this DSG-rich fraction and the cooking quality of pasta (0.63, P

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Two low molecular weight durum wheat (Triticum durum Desf) glutenins, DSG-1 and DSG-2 (durum-wheat sulphur-rich glutenin fractions) were isolated from two cultivars, Mondur of good technological quality and Kidur of poor technological quality. The glutenin fraction, composed mostly of DSG protein, was extracted using a low concentration of NA-tetradecanoate and then fractionted by by using molecular sieve chromatography (Bio-Gel P 30). The amino acid compositions and the N-terminal sequences of the pure DSG proteins were determined, and their hydrophobic characteristics, calculated on the basis of these data, showed that DSG-2 is more hydrophobic than DSG-1. The amino acid compositions of DSG-1 and DSG-2 were different. The N-terminal amino acids of DSG-1 and DSG-2 were also different but were identical for each of the two cultivars. In the case of DSG-1, in addition to the main chain a minor chain was found in which the first three amino acids of the main chain were missing. The minor chain represented about 30% int he DSG-1 of mondur and almost 50% in Kidur.