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.

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Copy-DNA clones have been obtained that distinguish eight messenger mRNAs, moderately abundant in the axes of the germinating soybean (Glycine max (L.) Merr.) seedling. These clones have been used to characterize the size of the mRNAs and to anlyze the accumulation of the mRNAs at different time points and in different parts of the axis during germination and early seedling growth. Three of the mRNAs accumulate to a substantial level by 9 h, a time point before either the beginning of growth or the accumulation of polyribosomes. Four other mRNAs reach a substantial level only at 24 h, a period when rapid seedling growth is occurring. Those mRNAs whose accumulation begins at 24 h were found only in the top (hypocotyl) half of the 24-h seedlings, while the remaining mRNAs were present also in the bottom half of the seedlings in different amounts. By 44 h, the bottom 0.5 cm of the seedlings, i.e., the region of meristematic growth, had little or none of the mRNAs, with the exception of one mRNA. These temporal and spatial observations indicate that many of the mRNAs are not involved simply in the general maintenance of ongoing cell proliferation, but that they may be related to differentiation during early seedling formation. Further, the early accumulating mRNAs may be functioning in regulating the onset of seedling growth.

Peptidyl proline hydroxylase inhibitors block the growth of cultured soybean (Glycine max) cells and bring about the disappearance of the major salt-extractable hydroxyproline-rich protein, the 33 kilodalton repetitive proline-rich protein (RPRP2). Three polypeptides of 28, 20, and 14 kilodalton that cross-react with an antibody to RPRP2 accumulate in the culture during steady-state growth. In the presence of the proline hydroxylase inhibitors, all of these repetitive proline-rich proteins disappear. These results indicate that the hydroxyproline-nch proteins play a role in cell growth, and that hydroxylation may regulate the steady-state level of at least one of these proteins by influencing its turnover.Hydroxyproline-containing proteins are found throughout the plant kingdom, generally associated with the extracellular matrix (9). Some of these proteins have been solubilized and characterized (12,17), and while morphoregulatory roles have been suggested, there is only limited supportive evidence (discussed in ref. 18). Considering that these proteins might be functionally dependent on the hydroxylation of their prolines, we have tested the effect of two specific inhibitors of prolyl hydroxylase (3, 16) in a soybean cell culture. We report here that the inhibition of peptidyl proline hydroxylation brings about the cessation of cell growth, accompanied by the disappearance from the culture of the major salt-extractable hydroxyproline-rich proteins, the RPRPs2 (2). These results suggest a role for hydroxyproline-rich proteins in regulating cell growth. MATERIALS AND METHODSAliquots of a soybean (Glycine max) cell culture, grown as previously described (2) Tris.HCl (pH 8), 2.5 mM NaHSO3 (0.5 mL per 5 mL medium). The cells were collected on a coarse scintered funnel, washed thoroughly with 10 mM Tris HCl (pH 8), 2.5 mM NaHSO3, frozen in liquid N2, and stored at -70°C. Growth of the culture was determined from the fresh weight of the cells obtained before freezing. To separate cell walls and cytoplasm, the frozen cells were broken in a polytron in five volumes of 0.25 M sucrose, 40 mm Tris.HCl (pH 8), 3 mM EDTA, 5 mm dithiothreitol, 0.5 mm p-toluenesulfonylfluoride. The cell walls were collected on a coarse-frit glass funnel (Millipore XX 1004700), and the cytoplasm was clarified by centrifuging for 1 h at l00,OOOg in a Spinco ultracentrifuge.The cell walls were washed several times with 10 mm Tris HCl (pH 8), 2.5 mm NaHSO3, and sonicated in 3 mL (per 0.6-0.7 g of initial cells) of 50 mm HCI, 0.2 M CaCl2, 2.5 mM NaHSO3. Extraction was continued for 3 h at room temperature, the insoluble residue was removed by filtration on nitrocellulose and the extract was neutralized with 1.2 M Tris base. Aliquots were run on SDS-16% acrylamide gels together with several levels of purified RPRP2 (4). The gels were transferred to Immobilon-P (Millipore Co.) in AMPSO buffer pH 9.8 (19) and probed with a rabbit polyclonal antibody (1--2000 dilution) made against the 33 kD RPRP (4). The procedure allowed the detection of 0.4 ...

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