Differentiation of Six Distinct Thioredoxins in Seeds of the Soybean

J. Plant Pbysiol. Vol.

146. pp. 385-392 {1995}

Differentiation of Six Distinct Thioredoxins in Seeds of the Soybean INGO ffiBERLEIN, MARKUS WOLF, LOUISIANNE MOHR,

and HARTMUT FOLLMANN

Fachbereich Biologie-Chemie der Universitat Kassel, Heinrich-Plett-StraBe 40, D-34109 Kassel, Germany Received October 21, 1994 . Accepted February 8, 1995

Summary

The thioredoxin profile of non-photosynthetic plant tissues, in particular of seeds, is much less known than that of green leaves. We have systematically fractionated and assayed the low molecular weight, heat-stable proteins present in extracts from soybean seeds by a variety of chromatographic and enzymatic methods. A multiplicity of six thioredoxins has been established, closely comparable to the pattern previously observed in soybean leaves (Haberlein, 1991). Purification to homogeneity of the six proteins was achieved by FPLC. Thioredoxin activities in vitro towards soybean chloroplast NADP-MDH, fructosebisphosphatase, NADPH thioredoxin reductase, and E. coli ribonucleotide reductase have been measured. Some of the seed thioredoxins exhibit higher molecular weights, stronger binding to anion exchange columns, and much lower specificity in enzyme assays than their counterparts isolated from leaves, suggesting the presence of unspecialized storage forms in the dry seed.

Key words: Glycine max (L.) Merr.; enzyme activation; seed proteins; thioredoxins. Abbreviations: FbPase = fructose-1,6-bisphosphatase; FPLC = fast protein liquid chromatography; MDH = malate dehydrogenase; NTR = NADPH thioredoxin reductase; TR = thioredoxin.

Introduction

The existence of multiple thioredoxins, first recognized in spinach leaves and in green algae fifteen years ago (Wolosiuk et al., 1979; Wagner et at, 1978), has been documented in a sizeable number of plant species (Berstermann et al., 1983; Vogt and Follmann, 1986; Hutcheson and Buchanan, 1979; Crawford et at, 1986). In contrast, bacterial and mammalian cells contain only one or two of the small, acid- and heatstable proteins. Despite these differences in distribution all thioredoxins possess the same active site formed by the amino acid sequence Cys-Gly-Pro-Cys (or, in few cases, CysAla-Pro-Cys) which enables them to transfer reducing equivalents through reversible cysteine-cystine redox changes in a wide variety of biochemical reactions (Holmgren, 1989). Interest in plant thioredoxins has originally focussed on their functions in chloroplasts (Wolosiuk and Buchanan, 1977). Thioredoxins m and f, acting as protein disulfide re© 1995 by Gustav Fischer Verlag, Stuttgart

ductases, mediate the light-dark regulation of an impressive number of enzymes engaged in carbon dioxide fixation (Buchanan, 1991), ATP production (Galmiche et al., 1990), chlorophyll synthesis (Kotzabasis et al., 1989), and in sulfate assimilation (Schwenn, 1989). However, thioredoxins also provide the reducing system for reduction of ribonucleotides to 2' -deoxyribonucleotides which is an extrachloroplastic process. Although the cytosolic thioredoxin-ribonucleotide reductase system has remained elusive in higher plants it could be clearly demonstrated in green algae (Scenedesmus obliquus (Turpin) Klitz) (Hofmann et al., 1985). Early reports of cytoplasmic leaf thioredoxins (Crawford et at, 1979) proved to be artifacts, but extrachloroplastic thioredoxins (termed thioredoxin h) have now been isolated from heterotrophic carrot cells, spinach roots and leaves Oohnson et at, 1987; Florencio et al., 1988). Since we identified specific thioredoxins in mammalian, yeast, and plant mitochondria (Bodenstein et at, 1989; Konrad, 1993; Konrad,

386

INGO fiXBERLEIN, MARKUS WOLF, LOUISIANNE MOHR, and HARTMUT FOLLMANN

Banze and Follmann, unpublished), however, extrachloroplastic thioredoxins cannot automatically be regarded cytosolie species. As thioredoxins are a highly significant family of plant proteins, it is imperative to obtain a thorough picture of their distribution, biochemical specificities, and developmental changes. Such information requires systematic fractionation of total tissue extracts and of intact plant cell organelles; moreover it is necessary to use several independent thioredoxin assays because the proteins possess no activity of their own and their specificities towards potential target enzymes cannot be predicted. The complete thioredoxin pattern of soybean (Glycine max (L.) Merr.) leaves has been described in this way (Haberlein, 1991). It comprises six different proteins, two of which are located in chloroplasts and two in the mitochondria. Plant seeds, without photosynthetic functions, could possess a smaller number of thioredoxins. Indeed, in wheat and soybean seeds only three or four thioredoxins have been found (Berstermann et aI., 1983; Vogt and Follmann, 1986), but a complete inventory of thioredoxins in plant seeds using high-resolution protein fractionation has not been compiled. In this paper we describe the total thioredoxin profile of soybean seeds which, for the first time, permits comparison between the thioredoxins present in leaves and seeds of the same plant. It is shown that seed thioredoxins are as numerous as their counterparts in leaves.

Materials and Methods

General All chemicals and reagents were of highest purity available and were obtained from Merck, Biomol, or Serva. Electrophoresis on SDS-polyacrylamide (12%) gels was performed in a Tris/Tricine (pH 8.3) buffer system as described (Schagger and von Jagow, 1987). Soybeans (Glycine max (L.) Merr. cv. Maple Arrow) were obtained from Kupper Mitteldeutsche Samen GmbH, Eschwege, and were used within 1 year. Soybean plants were grown in the departmental greenhouse, and spinach (Spinacia oleracia L.) was purchased at local markets. Heterotrophic soybean cells were cultured in B5 medium (Gamborg et a!., 1968) for 10 days in the dark.

Enzymes Chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) and fructose-l,6-bisphosphatase (EC 3.1.3.11) were freshly prepared from soybean and spinach leaves as described below. Ribonucleotide reductase from E. coli (EC 1.17.4.1) and NADPH thioredoxin reductase (EC 1.6.4.5) from soybeans were purified by published procedures (Brown et a!., 1969; Berstermann et a!., 1983; Wolf, 1986). Fructose·l,6·bisphosphatase: Leaves (100 g) from 4-8week old soybean plants were homogenized with 100 mL cold extraction buffer (80mM KH zP0 4, pH 7.0) in a Waring blender. The homogenate was passed through three layers of Miracloth and centrifuged for 20 min at 38,700 x g. The pH of the supernatant was adjusted to 4.S by addition of acetic acid. The precipitated protein was collected by centrifugation (5 min at 38,700 x g) and redissolved in 100 mL buffer (25 mM HEPES, 0.25 mM EDTA, pH 7.6). Insoluble material was removed by centrifugation. Ammonium sulfate was added to 50 - 90 % saturation, the resulting precipitate was redissolved in N a-

acetate buffer (50 mM, pH 5.5), and the protein fractionated on a Sephadex GI00 column. Further purification was achieved by DEAE cellulose (Whatman DE 52) anion exchange chromatography. FbPase eluted at O.SM NaCI in a linear gradient of 0-lM NaCI in 200 mL of column buffer (50 mM, Na-acetate, pH 5.5). The resulting FbPase preparation had a protein concentration of 0.13 mg/ mL and a purity of about 80 %. Isolation of FbPase from spinach leaves was done by the same procedure except that the DEAE cellulose column was operated in a gradient of O-O.5M NaC!. Spinach FbPase, eluted at 0.35M NaC!, had a protein concentration of 0.16 mg/ mL and was about 80 % pure. NADP Malate dehydrogenase: Soybean leaves (100g) from 4 - 8 week old plants were homogenized with 100 mL extraction buffer (100 mM KH zP04, pH 7.0, 0.1 % mercaptoethanol) in a Waring blender, and the homogenate was passed through three layers of Miracloth. Cell debris were removed by centrifugation (20 min; 38,700 x g). Ammonium sulfate was added to 3S - 80 % saturation, the precipitate was collected by centrifugation (20 min; 38,700 x g), and redissolved in a minimum volume of Tris-HCI buffer (100 mM, pH 7.9) containing 100 mM NaCI. Insoluble material was removed again by centrifugation, and the solution was applied onto a Sephadex G 100 column equilibrated in the same buffer. The enzyme, eluted in the void volume, had a protein concentration of 0.42 mg/ mL and was about 40 % pure. It was strictly specific for oxaloacetate and NADPH and strongly activated by thioredoxin. NADP-MDH from spinach was isolated from an extract of 500 g leaves by the same procedure. It was purified further on a column of Red Sepharose and eluted at 0.5 M NaCI concentration in a linear gradient of 0-2M NaCI in 200mL column buffer (20mM' KH zP0 4; pH 7.0). The protein concentration of this preparation was 1.65 mg/ mL and the purity about 90 %.

Purification of thioredoxins from soybean seeds All steps were carried out at 4°C. Seeds were finely ground in 100 g batches and the flour was extracted under stirring for 2 hours in 1 L of 100 mM Tris-HCI buffer, pH 7.5, containing 1 mM EDTA. Other tissues (cf. Table 2) were extracted in an analogous way. The material was passed through a cloth and the extract centrifuged for 20 min at 13,700 x g. The supernatant was subjected to heat denaturation (3 min at 70°C) and acid treatment (PH 4.6; 30 min), and precipitated proteins were removed by centrifugation (20 min; 38,700 x g). Ammonium sulfate (90 % saturation) was added to the supernatant, and the precipitate was collected by centrifugation. The pellet was redissolved in 150 mL 20 mM ammonium acetate buffer, pH 8.6, containing 2 mM EDTA, and dialyzed against 2 x 5 L of the same buffer. This fraction contains all thioredoxin proteins. Partial fractionation by ion exchange chromatography: The dialyzed fraction was chromatographed on a DEAE cellulose column (Whatman DE 52; 8 x 2.8 em) equilibrated with 20 mM ammonium acetate/2 mM EDTA (pH 8.6). Three thioredoxin activities, TR I, TR II and TR III, were obtained. TR I was found in the void volume whereas TR II and TR III separated in a linear salt gradient of 0-0.3M NaCI in 400mL of column buffer (flow rate, SOmLlh). In case of an unsatisfactory separation between TR II and TR III the material was rechromatographed on a column of Fractogel TSK DEAE 65 SW (Merck; 2x2.8cm), equilibrated with SOmM TrisHCI buffer, pH 8.6. The three thioredoxin fractions were purified further on CM cellulose (Whatman CM 52; 5 x 2.8 em) equilibrated with 10 mM sodium acetate buffer, pH 4.6. TR I, TR II and TR III were bound and eluted at 200 mM NaCI in a linear gradient of 50-250 mM NaCI in 400 mL of column buffer (flow rate, 30 mLlh). Active fractions were neutralized with 1 M NaOH and concentrated by ultrafiltration over an Amicon YM 5 membrane. Thioredoxins I, II, and III

Soybean Seed Thioredoxins were finally passed over a Sephadex G50 column (140 x 1.5cm) equilibrated with 50 mM Tris-HCI buffer containing 100 mM NaCI, pH 7.5, at a flow rate of 10 mL/h. Active fractions were dialyzed against 10 L of Mono Q column buffer. FPLC Chromatography: All soybean thioredoxins were purified to homogeneity on the Mono Q HR siS column of a Pharmacia FPLC system. The column was equilibrated with 20 mM Tris-HCI buffer, pH 8.3, and eluted with a linear gradient of 0-200 mM NaCI in 20 mL of column buffer. Fractions of 0.5 mL were collected at a flow rate of 0.5 mL/min. When a second Mono Q chromatography of the resolved thioredoxin species did not yield homogeneous protein, additional purification was performed on a Mono S HR sis column equilibrated with 50 mM sodium acetate buffer, pH 4.6. Thioredoxins eluted from Mono S in a linear salt gradient of 0-200mM in 20mL column buffer. Fractions of 0.5mL were collected at a flow rate of 0.5 mLImin. Analytical resolution of the complete thioredoxin pattern: Ground soybeans (100 g) were extracted and the extract subjected to heat and acid treatment as described above. The unresolved thioredoxin mixture was fractionated on a Sephadex G50 column followed by chromatography on CM cellulose and by a second Sephadex G50 column. The highly enriched, total thioredoxin sample was analyzed by FPLC on a Mono Q column equilibrated and operated in 20mM Tris-HCI buffer, pH 8.0, plus a 0-200mM NaCI gradient.

Enzyme assays Fructose-l,6-bisphosphatase: Thioredoxin-dependent activation of FbPase was performed by 30 min incubation at 30°C in a mixture (0.5 mL) containing 200 mM Tris-HCI, pH 7.9, 1 mM dithiothreitol, 5 mM MgS0 4 , 100 ilL of the specified FbPase fraction, and variable amounts of thioredoxin. Enzyme activity was then measured after addition of 50 ilL 60 mM fructose-1,6-bisphosphate in assay buffer (200 mM Tris/CI, pH 7.9) and incubation for 10 min at 30°C. The reaction was stopped with 50 ilL of 72 % trichloroacetic acid, precipitated protein was removed by centrifugation, and inorganic phosphate was determined photometrically in 0.5 mL of the reaction mixture (Taussky and Shorr, 1983). NADP-malate dehydrogenase: Thioredoxin-dependent activation of NADP-MDH was accomplished within 30 min incubation at 30°C. The incubation mixture (0.8 mL) contained Tris-HCI buffer (200 mM, pH 7.9), 5 mM dithiothreitol, 100 ilL of the specified enzyme preparation, and varying amounts of thioredoxin. Enzyme activity was measured spectrophotometrically after addition of

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Three thioredoxin fractions I - III were previously identified among the heat-stable, soluble proteins of a soybean flour extract (Berstermann et aI., 1983) and were numbered according to their elution from an anion exchange column. Because they lack specificities in activating thioredoxin-dependent enzymes in vitro (see below), and direct assignment of their intracellular location in a dry seed is not feasible, we retain the neutral numbering scheme in our present analysis. Following heat (3 min, 70°C) and acid (30 min, pH 4.6) treatment, which proved necessary to reduce the high amount of protein (30 - 35 g) present in an extract from 100 g soybeans, chromatography on DEAE cellulose is a superior method to separate the three active fractions; thioredoxin I passes through the column whereas II and III are resolved in an NaCI gradient (Fig. 1 a). In case of incomplete separation of proteins II and III it is of advantage to rechromatograph on a different anion exchanger, Fractogel TSK-DEAE, which binds thioredoxin III but not protein II (Fig. 1 b). Further purification of the three thioredoxin fractions can be achieved by CM cellulose chromatography and gel filtration on Sephadex GSO columns but additional thioredoxin species are not observed in these steps. Considering the multitude of six thioredoxins in soybean leaves (Haberlein, 1991) soybean seed proteins I, II and III

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were subjected to high resolution FPLC on a Mono Q anion exchange column. Indeed thioredoxin fractions I and II were resolved into several thioredoxin activities by this technique (Fig. 2 a, b). Protein I was separated into TRIa and TRIb, and protein II was resolved into three species, TRlIa, TRlIb, and TRlIc. Fraction III remained unchanged (Fig. 2 c). Homogenous thioredoxin preparations were then obtained by repeating the Mono Q chromatography. Alternatively, the individual thioredoxins could be subjected to FPLC-Mono S cation exchange chromatography for final purification (data not shown). A yield of about 50 ~g of homogeneous thioredoxin species was achieved in this way. It is not possible to estimate the original amounts and recoveries of the individual proteins because they cannot be differentiated in an unresolved mixture. The differentiation of no less than six thioredoxins in an extract from non-photosynthetic plant tissue, never encountered before, raised the question of artefacts. When the preparation of the soybean seed thioredoxins was carried out in the presence of protease inhibitors (0.5 mM phenylmethylsulfonylfluoride, 2 mM aminocaproic acid, and 2 mM benzamidine-HCI), virtually unchanged chromatographic profiles were obtained. In another experiment designed to eliminate chromatography artefacts, the total thioredoxin content of soybean seeds was enriched, but not resolved, by heat and acid treatment, CM cellulose, and Sephadex chromatography. The individual proteins were separated in one

final FPLC run at pH 8.0 and their activities assayed in parallel with two different indicator enzymes, NADP-MDH and alkaline fructosebisphosphatase (Fig. 3). All six thioredoxins la-III observed in the original protocol were also identified in the protein profile eluted between 85 and 190mMNaCl.

Properties of seed thioredoxins The purified soybean seed thioredoxins were analyzed by denaturing polyacrylamide gel electrophoresis (Fig. 4). They vary in molecular mass from 10,000 to 16,000 Da (protein la: 16,000; Ib, IIa, and lIb: 15,000; thioredoxins IIc and III: 11,000 and 10,000 Da, respectively). Proteins IIc and III exhibit the molecular weight characteristic of thioredoxins from most sources (including soybean leaf thioredoxins; Haberlein, 1991) whereas the other four seed protein are distinctly larger. All other properties of the seed thioredoxins such as heat stability (70°C) and acidic isoelectric points (4.5 - 5.7) are also within typical limits.

Reactivity in enzyme assays The specific functions of multiple thioredoxins in plant seeds are not obvious and have not been analyzed. We, therefore, measured the activities of soybean seed thioredoxins in four different reactions, viz. to activate two chloroplast en-

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and Follmann, 1979) is not available in purified form but the enzyme from Escherichia coli is known to interact with any heterologous thioredoxin. Thioredoxin from E. coli served as reference in these assays. The data are summarized in Table 1. Inspection of thioredoxinlenzyme interaction in the vertical columns reveals that all soybean seed proteins possess similar activity with each of the five plant enzymes; individual differences do not exceed 2- to 3-fold. An exception from this unspecific behaviour is the good reactivity of thioredoxin III with ribonucleotide reductase which reacts poorly with the other seed proteins.

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The proportions of the different thioredoxins present in the soybean should reflect physiological functions, but their differentiation and quantification in a total extract is not feasible. However, the initial separation of thioredoxin fractions I, II, and III by DEAE cellulose chromatography (Fig. 1 a) is highly reproducible, and the specific activities of all six proteins towards NADPH-MDH and FbPase in vitro are similar (Table 1). Therefore, the anion exchange chromatography step permits a semiquantitative estimate of the presence and amount of the various thioredoxins in extracts from seeds, leaves, and other heterotrophic or phototrophic cells (Berstermann, 1984). Protein extracts prepared from seeds, etiolated and green seedlings, leaves, roots, and suspension culture cells were analyzed in parallel in this way (Table 2); the thioredoxin fractions present in chloroplast or mitochondriallysates (Haberle in, 1991; Konrad, 1993) are given for comparison . It is evident that thioredoxins I, identified as chloroplast proteins in leaves, predominate in photosynthetic tissues whereas protein III is the most prominent thioredoxin in seeds and heterotrophically growing cells. Thioredoxin II, of mitochondrial origin, is always present in intermediate, fairly constant proportion.

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Fig.3: FPLC Analysis of an unresolved mixture of soybean seed thioredoxins. Cf Materials and Methods for experimental details. a: Thioredoxin activities determined by chloroplast NADP-MDH (.1A)4Q· min -I); b: fructosebisphosphatase activation assays (phosphate liberation; .1Aow); c: protein absorption at 280 nm. zymes, NADP-MDH and alkaline FbPase, to serve as reductant in the dithiol-requiring ribonucleotide reductase system, and as substrate of NADPH-thioredoxin reductase (NTR). Since it has been our experience that activity determinations of plant thioredoxins are reliable only using homologous enzymes (Haberlein et aI., 1985) the first two enzymes were prepared from soybean leaves as described in the Experimental section, and NTR was isolated from soybean seeds as described earlier (Wolf, 1986). The commonly used spinach chloroplast enzymes were employed for comparison. Ribonucleotide reductase from soybean (Hovemann

Discussion

Plant seeds or heterotrophic plant cells without light-regulated functions have been thought to possess less complex thioredoxin patterns than leaves and to provide a source for extrachloroplastic thioredoxins. In fact, the number of 1- 3 thioredoxins isolated from wheat, soybean and castor bean seeds (Berstermann et aI., 1983; Vogt and Follmann, 1986; Marcus et aI., 1991), heterotrophic algae and carrot cell cultures (Langlotz and Follmann, 1987, Johnson et aI., 1987), and spinach roots (Florencio et aI., 1988), is smaller than the multiplicity of 4 - 6 thioredoxins found in green algae and leaf extracts (Crawford et al., 1986; Florencio et al., 1988; Haberlein, 1991). On the other hand seeds and seedlings contain various precursor forms of green plastids which have not been analyzed for thioredoxin content. The discrepancy between one thioredoxin in potato tuber mitochondria and two proteins in leaf mitochondria (Bodenstein et aI., 1989; Konrad, 1993) further complicates the picture. An analysis

390

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INGO HABERLEIN, MARKUS WOLF, LOUISIANNE MOHR, and HARTMUT FOLLMANN

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Table 1: Reactivity of soybean seed thioredoxins. The enzymes' were combined with identical amounts of thioredoxin (5 J,tg) and assayed under identical conditions, adjusted individually to ensure a linear range of thioredoxin activation. Differences in absolute activity of the six enzymes are not relevant in this comparison. Thioredoxin

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NADP-MDH: NADP-malate dehydrogenase; FbPase: fructosebisphosphatase; RRase: ribonucleotide reductase; NTR: NADPH-thioredoxin reductase. The unresolved thioredoxin fractions I and II, respectively, were used in these assays.

Table 2: Percentage of thioredoxin fractions I - III in extracts from different soybean tissues and cells. The fractions were obtained by DEAE cellulose chromatography and assayed with spinach NADPMDH under identical conditions. The thioredoxin content of isolated chloroplasts and mitochondria has been determined separately (Haberlein, 1991; Konrad, 1993). Source

Total activity limol NADPH/min

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of the complete thioredoxin pattern in seeds and leaves of the same plant is obviously desired. The identification, and purification to homogeneity of all thioredoxins present in a plant extract is not trivial, given their structural similarities, different intracellular amounts, and lack of individual, specific in vitro assays. We have relied on advanced anion exchange methods in our present analysis of soybean extracts. It demonstrates, for the first time, that a

seed contains as many different thioredoxins as a green plant. While the fractionation of soybean proteins by conventional chromatography and preparative electrophoresis (Berstermann et al., 1983) remained insufficient, the optimized, rapid work-up scheme produced consistent results, regardless whether the six thioredoxins were resolved in separate FPLC runs (Fig. 2), or their mixture was fractionated in one step (Fig. 3). Other chromatography media were tried but did not generate further subfractionation. The hexadic thioredoxin profile of soybean seeds is, therefore, considered complete and devoid of artefacts. The similarity of chromatographic thioredoxin patterns in seed and leaf extracts of soybean (Hiberlein, 1991) is remarkable. With the exception of protein III, however, the seed thioredoxins require 10-40 mM higher NaCI concentration for elution from the anion exchange column. Moreover, thioredoxins la, Ib, IIa and lIb (Mr = 15 -16 kDa) are larger than the leaf proteins (Mr = 12 -12.5 kDa) which elute in the same order. In leaves thioredoxins la and Ib are unambiguously identified as chloroplastic, and proteins lIb and IIc as mitochondrial thioredoxins (Haberlein, 1991; Konrad, 1993). We hypothesize that these leaf thioredoxins are stored in the seed organelles as precursor molecules containing about 15 - 25 additional amino acids (not necessarily identical with the polypeptide precursors synthesized on cytoplasmic ribosomes). The lack of specificity of seed thioredo-

Soybean Seed Thioredoxins xins towards homologous chloroplast enzymes in vitro established above (Table 1) is in accord with a role as silent, unspecialized storage forms: The observed reactivities merely reflect the exchangeability of thioredoxins and thioredoxinactivated enzymes long known in in vitro assays (Wagner et aI., 1978). In contrast, the individual leaf thioredoxins exhibit higher specificities and complex kinetics in homologous combination with soybean leaf enzymes (Vogeler and Hiberlein, unpublished). Soybean thioredoxin III is clearly a cytosolic protein because it is the predominant thioredoxin in seeds, etiolated seedlings, roots, and heterotrophic cell cultures, present in lower amount in green seedlings and leaves, and not found in leaf chloroplasts and mitochondria (Table 2). Its abundance in the proliferative tissues is in accord with a function in deoxyribonucleotide biosynthesis. Ribonucleotide reductase has been demonstrated in legume seeds and cell cultures (Hovermann and Follmann, 1979), but the plant enzyme is unstable and attempts to measure activity in combination with soybean thioredoxins in vitro have failed. However, the reactivity of thioredoxin III with a stable bacterial ribonucleotide reductase (Table 1) is strong evidence for its physiological role. A function of thioredoxin/NADPH thioredoxin reductase during seed germination was predicted (Suske et al., 1979) and recently demonstrated in vitro (Kobrehel et aI., 1991, 1992) in the reductive mobilization of storage proteins and inactivation of protein inhibitors. The specificity of these reactions with respect to thioredoxins and target proteins has not been fully established. Prospectively, purified soybean seed thioredoxins will also allow to study physiological interactions with the legumin and glycinin storage proteins of Glycine max.

Acknowledgements

This work has been supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 305 (Okophysiologie), and by Fonds der Chemischen Industrie. We thank Drs. Andrea Berstermann and Claus Bornemann for their contributions.

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