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. Endo The seed is the only tissue for which the NADP/thioredoxin system has been ascribed physiological activity in plants. Thioredoxin h reduces members of several different soluble seed proteins-thionins, a-amylase, and trypsin inhibitors (11, 14)-and also reductively activates an enzyme of carbohydrate metabolism (PPi fructose-6-P, 1-phosphotransferase, or PFP) (13). The results (14) suggest that the inhibitor proteins, long known to be active in bioprotection, may function within the seed to link thioredoxin to the regulation of yet-to-be identified target enzymes (cf. 9, 16, 21).The question arises as to whether thioredoxin can reduce other types of seed proteins. Quantitatively, the most important group is comprised of storage proteins, which account for up to 80% of the total protein of the seed (12,20). In the case of plants such as cereals, these proteins are insoluble in aqueous solutions and are chemically inert until they are reduced. It is not known how these proteins are mobilized during germination, and a physiological agent capable of their reduction has not been described.To help fill this gap, we have undertaken a study with wheat, a cereal with well-characterized seed proteins. We now report that representatives of the major wheat (Triticum durum) storage proteins-the gliadins and glutenins-are specifically reduced by thioredoxin. The results provide evidence that the NADP/thioredoxin system functions in the reduction of the principal seed proteins, thereby increasing their proteolytic susceptibility and making amino acids (nitrogen and sulfur) available during germination. Taken together with our recent work (14), the new findings suggest that thioredoxin functions as a signal to enhance metabolic processes associated with seed germination. A preliminary account of this work has been published (31). MATERIALS AND METHODS Plant MaterialSeeds and semolina of durum wheat (Triticum durum, Desf. cv Monroe) were kind gifts of Dr. K. Kahn. Germination of Wheat SeedsTwenty to thirty seeds were placed in a plastic Petri dish on three layers of Whatman No. 1 filter paper moistened with 5 mL of deionized water. Germination was carried out for up to 4 d at room temperature in a dark chamber. Plant Physiol. Vol. 99, 1992 Reagents/Fine Chemicals Biochemicals and lyophilized coupling enzymes were obtained from Sigma. Escherichia coli thioredoxin and NTR were purchased from American Diagnostica, Inc. (Greenwich, CT). Wheat thioredoxin h and NTR w...
Three forms of pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) were purified from both green and red tomato (Lycopersicon esculentum) fruit: (a) a classical form (designated 02) containing a-(66 kilodalton) and ,B-(60 kilodalton) subunits; (b) a form (01) containing a ,-doublet subunit; and (c) a form (Q0o) that appeared to contain a ,B-singlet subunit. Several lines of evidence suggested that the different forms occur under physiological conditions. Q2 was purified to apparent electrophoretic homogeneity; Q1 and Q0 were highly purified, but not to homogeneity. The distribution of the PFP forms from red (versus green) tomato was: Q2, 29% (90%); 01, 47% (6%); and Q0, 24% (4%). The major difference distinguishing the red from the green tomato enzymes was the fructose-2,6-bisphosphate (Fru-2,6-P2)-induced change in Km for fructose-6-phosphate (Fru-6-P), the 'green forms' showing markedly enhanced affinity on activation (Km decrease of 7-9-fold) and the 'red forms' showing either little change (Q0o, Q1) or a relatively small (2.5-fold) affinity increase (02). The results extend our earlier findings with carrot root to another tissue and indicate that forms of PFP showing low or no affinity increase for Fru 6-P on activation by Fru-2,6-P2 (here 01 and Qo) are associated with sugar storage, whereas the classical form (02), which shows a pronounced affinity increase, is more important for starch storage. from most plant sources is highly stimulated by Fru-2,6-P2 (1, 2, 5, 6, 8-10, 15-18, 20, 21), the enzyme from leaves of CAM plants responds only sluggishly to this activator (5,8,10). In another case (wheat seedlings), PFP activity has been reported to be associated with two structurally different enzyme forms (21). Finally, in a recent study (18), our laboratory has identified in carrot roots a form of PFP that differs both kinetically and structurally from other forms of the enzyme studied to date.As a result of this unusual kinetic and structural diversity, we have considered the possibility that the properties of PFP depend on the functional nature of the parent tissue (18). That is, the form of PFP present in one tissue with a given metabolic function may differ significantly from the enzyme in a tissue with another function. We have put this idea to a further test using pericarp of tomato fruit-a tissue known to change from a starch-storing to a sugar-storing function during ripening (7,14). The results indicate that, while occurring in analogous enzyme forms, the PFPs from green and red fruit show a pronounced difference on activation by Fru-2,6-P2. The activated green forms are characterized by a marked increase in affinity for Fru 6-P, whereas the red forms show either little or no change in this connection. A preliminary account of this work has been published (19
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