Purification and Characterization of a Soybean Leaf Storage Glycoprotein

Abstract

Removing the pods from soybean (Glycine max [L.] Meff. cv Wye) plants induces a change in leaf function which is characterized by a change in the leaf soluble protein pattern. The synthesis of at least four polypeptides (-27, 29, 54, and 80 also demonstrated a marked increase in the accumulation of protein within the vacuoles of these cells following pod removal. Therefore, the polypeptides that accumulate following depodding may be localized in these cells.The purpose of this study was to further characterize these polypeptides and to purify one or more to obtain antiserum for quantitation and cellular localization work. This paper describes the purification and antiserum production to a glycoprotein apparently composed ofa 27 and 29 kD polypeptide. In addition, quantitation of this protein with development and further characterization of its accumulation are presented. MATERIALS AND METHODS Plant Material. Soybean (Gycine max [L.] Merr. cv Wye[determinate]) plants were grown as previously described (12). Depodding was initiated 1 week after flowering and was repeated at weekly intervals.Protein Purification. Trifoliate leaves were harvested from the main stem of plants which had been depodded for 4 to 5 weeks. The leaves were extracted in 20 mM Tris-HCI buffer (pH 7.6) containing 4 mM DTT and 1 mM EDTA (4 ml: 1 g of tissue) at 4°C using a Waring Blendor. The homogenate was centrifuged at 30,000g for 20 min, and the supernatant fractions were decanted and combined. Saturated (NH4)2SO4 was added to the supematant fraction to bring it to a concentration of 2.0 M and, after standing for 30 min on ice, the solution was centrifuged at 12,000g for 10 min. The precipitate was discarded and the supernatant solution was brought to 2.8 M (NH4)2SO4. After centrifugation, the precipitate was dissolved in 100 mm acetate buffer (pH 5.6) containing 0.9% NaCl. This solution was applied to a Con A-Sepharose column (1 x 5 cm) equilibrated with the resuspension buffer. The column was washed with buffer until no more protein was eluted; then 50 mM l-O-methyl-a-D glucopyranoside in the same buffer was added to elute the bound protein. The fractions containing the bound protein were then applied to a Sephacryl S-200 column (5 x 105 cm) equilibrated with 0.9% NaCl in 20 mm Tris-HCl buffer (pH 7.6). One major peak (A280) was obtained, and the fractions from the center of this peak were combined and concentrated for antiserum production and standardization of the quantitative radial immunodiffusion assay.Production of Antiserum. The purified protein was emulsified in complete Freund's adjuvant. Mice (C57/BL6) were immunized initially by footpad injection with 50 ,ug protein/mouse. Subsequent boosts after 2 and 3 weeks were done intraperitoneally using 10 and 5 gg protein, respectively, plus 1 mg of Alhydrogel/mouse. The mice were then bled 1 week later by retroorbital puncture to obtain antiserum. Thereafter, the mice were given boosts of 1 to 1.5 ,ug protein every 2 to 3 weeks and were bled 1 week after each boost for col...

Characterization of a Soybean Leaf Protein That Is Related to the Seed Lectin and Is Increased with Pod Removal

“…), a glycoprot-ein has also been identified that accumulates markedly when reproductive sink demand is lowered by depodding (26). However, soybean VSP3 appears to be distinct from the major seed lectin of soybean (23,27). Peptide sequences derived from nucleic acid sequences ofthe VSP subunits indicate only limited homology with soybean agglutinin (23), and no hemagglutinating activity has been reported for the purified protein.…”

“…Some of these glycoproteins are immunologically similar to the seed lectins isolated from the same species (6,27), but others appear to be immunologically distinct from any seed protein (for reviews, see refs. 6, 12, 14).…”

“…It is now well established that vegetative tissues contain glycoproteins that have similarities to seed storage proteins (6,27). Some of these glycoproteins are immunologically similar to the seed lectins isolated from the same species (6,27), but others appear to be immunologically distinct from any seed protein (for reviews, see refs.…”

Cell-Wall Proteins Induced by Water Deficit in Bean (Phaseolus vulgaris L.) Seedlings

“…Vol. 107,1995 proteins (Mason et al, 1988;Mason and Mullet, 1990) revealed that they were identical with the soybean leaf VSPs (Wittenbach, 1983).…”

The Expression Profile of Lectin Differs from That of Seed Storage Proteins inCastanea crenataTrees

“…17) Soybean VSPs accumulate transiently in developing organs, especially immature leaves or pods, and then decrease in content with seed maturation. 18) Increasing numbers of reports of VSPs in various plants have indicated that in woody perennials they relate to dormancy and respond to outer stimuli, such as photoperiod, chilling-accumulation, and wounding, as well as to the effects of sugar, nitrogen, and auxins.…”