A cDNA clone that hybridizes to an mRNA (lA10) that accumulates to a substantial level in the axis of the germinating soybean seed was sequenced. The amino acid sequence of the clone indicates an almost perfect repeat of Pro-Pro-Val-Tyr-Lys resulting in a protein containing 40% proline and lacking serine and histidine. On the likelihood that such a protein might be a hydroxyproline-rich cell-wall-glycoprotein (HRGP), cell walls of a soybean cell culture were extracted by procedures used to obtain soluble basic cell-wall glycoproteins, and the proteins were fractionated and purified. A 33-kDa protein (and possibly a 28-kDa protein) was obtained that has an amino acid distribution similar to that of the cDNA clone. The protein lacks histidine and serine and contains 20% hydroxyproline and 20% proline. The HRGP is thus distinct both in its amino acid content and in its pentameric repeat of Pro-Pro-Val-Tyr-Lys, with half of the prolines being hydroxylated. (4) was maintained on a 14-day cycle with a 1:12 dilution of cells (7 ml and 77 ml of medium in a 250-ml flask) every 2 weeks.In the preparation described in Results,25 ,uCi (1 Ci = 37 GBq) of [14C]proline was added to 8-day cells, and the cells were allowed to continue growing for 4 hr, after which time they were filtered on a coarse scintered funnel and frozen in liquid N2. In more recent preparations, 8-to 9-day cultures have been collected directly without labeling and the purification steps have been monitored solely by following the absorbance at 280 nm. Frozen cells (4.6 g) were homogenized in a Polytron in 0.25 M sucrose/3 mM EDTA/40 mM Tris-HCI, pH 8/0.5 mM phenylmethylsulfonyl fluoride/5 mM dithiothreitol (2.2 ml/g), and centrifuged for 2 min at 750 x g. The pellet was recentrifuged for 10 min at 23,500 x g and then washed one time in 14 ml of 5 mM potassium phosphate (pH 6.8) and six times with 15 ml of deionized water, centrifuging each time for 2 min at 750 x g. The pellet was extracted twice with 12 ml of 0.2 M CaC12 (5, 6), each extraction lasting 45 min. After centrifuging for 5 min at 2000 x g, the supernatant was adjusted with 68% trichloroacetic acid to a final concentration of 10% trichloroacetic acid, and the suspension was kept at 0C overnight. After centrifuging for 15 min at 24,000 x g, the supernatant was dialyzed against five changes of water over a 5-hr period and then overnight against 25 mM Tris HCI (pH 8.0). The dialyzer tubing had a Mr 3000 cutoff (Thomas 3787-H45). A typical preparation yielded 1.9 mg of protein based on 4 A280 units for a solution of 1 mg/ml, a figure calculated from the presence of 13 mol % tyrosine (see Table 1) in comparison with bovine serum albumin, which has 3.3% tyrosine (7) and 1 A280 unit for a solution of 1 mg/ml. The sample was chromatographed on a column of CM-cellulose (1.2 x 10 cm) equilibrated with 25 mM Tris-HCl (pH 8.0) and eluted with 100 ml of a linear gradient of 0.4 M NaCl in the same buffer. The 33-kDa protein, eluting at ""0.14 M NaCI, was pooled (tubes 17-27 in Fig. 3), concentra...

We have resolved and analyzed two proline-rich proteins isolated from the walls of soybean cells in culture. The proteins are similar in amino acid content, containing 20% proline, 20% hydroxyproline, 20% lysine, 16% valine, 10% tyrosine, and 10% glutamate. The proteins undergo a rearrangement or a limited cleavage in dilute NaOH, but are otherwise remarkably stable to a high concentration of alkali. We have cloned and sequenced a cDNA from soybean axes germinated for 31 hours (1A10-2) coding for a protein that closely corresponds in its amino acid content to that of the proline-rich proteins. The cDNA sequence predicts a decameric repeat of Pro-Pro-Val-TyrLys-Pro-Pro-Val-Glu-Lys. Consequently, this class of proteins is referred to as repetitive proline-rich proteins, i.e., RPRP2 and RPRP3. We have also analyzed RNA gel blots with probes that discriminate between the new cDNA clone and a related cDNA previously reported [SbPRPl;Hong, Nagao, and Key (1987). J. Biol. Chem. 262, 8367-83761. Messenger RNAs from young seedlings and from soybean suspension cultures correspond primarily to the new RPRP clone (1A10-2), whereas the predominant mRNA accumulating later in the roots corresponds to SbPRPl.

Siumtmt77ary. The kinetics of development of proteini-synthesizing capacitv in the imbibing wheat embryo, were sttudied both in vivo and in vitro. During the first 30 minutes of imbibition protein-synthesizing capacity rises rapidly, lagging about 10 minutes behind water uptake. This rise in synthesizing capacity is accompanied by an increase in polysome content. As imbibition continues, both protein-synthesizing capacity and polysome content increase. With embr,-os from aged seed, the rate of protein synthesis is initially limited by another, presuimably nonribosomal, reaction.An earlier report (4) concltuded that seed germination is accompanied by an increased capacity for protein synthesis. This conclusion was based primarily on the observation that ribosomes isolated from dry (unimbibed) Material and MethodsIn zizo Incorporation. Assay 1. Wheat embryo samples (200 mg) were imbibed for the desired times and then transferred to a test tube where they were incubated in 2.5 ,umoles potassium phosphate (pH 6.0), 40 jig chloramphenicol, 5.1 m,umoles L-leulcine-'4C (0.25 jucuiries) and water to 0.53 ml.The volume of incubation medium was such that the embryos were submerged. After incubation, 200 nmoles of L-leucine-_2C were added; free radioactivity was removed by washing with water, and the embryos were grauun(d to a homogeneous sUi'-pension in 5 % trichloroacetic acid containing 200 ,umoles L-leuicine-_2C. An appropriate aliquot of the stuspension was centrifuged; an aliquot of the supernatant was counted in Bray's solution (1) (trichloroacetic acid soluble). Another aliqulot of the suspension was centrifuged, resuspended in 5 % trichloroacetic acid, and heated for 15 minuites at 90°. After cooling for 10 minutes in ice, the precipitate was collected on a membrane filter, washed with 5 % trichloroacetic acid and counted in toluene-PPO-POPOP (trichloroacetic acid insoluble).

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