ABSTRACrMajor radish (Raphanus sativus L. cv National) proteins synthesized at the beginning of germination have been characterized by their migration in two-dimensional electrophoresis.The use of 15-minute labelings shows that these proteins are encoded by stored mRNA. phoresis patterns of polypeptides synthesized in vivo or in vitro (10), these authors have shown the presence of a specific set of mRNAs which accumulate during late embryogenesis and disappear during early germination. This work is presently the only one suggesting that some stored mRNAs code for proteins playing a specific role in early germination.We have previously detected the presence of stored mRNA in dry radish embryos and analyzed their life time (6,7). As a first step in the characterization ofproteins encoded by stored mRNA in radish, we have analyzed by two-dimensional electrophoresis the polypeptides synthesized in embryos during the first 15 min of germination and in an in vitro translation system directed by stored mRNA. These polypeptides have been compared with those synthesized during late embryogenesis and late germination and with those present in dry embryos.In this report, we show that the polypeptides synthesized during the first 15 min of germination are encoded by stored mRNA and can be classified into two sets. MATERIALS AND METHODS

The methylation pattern of radish Raphanus sativus nuclear rDNA has been investigated using the Hpa II, Msp I, and Hha I restriction enzymes. The presence of numerous target sites for these enzymes has been shown using cloned rDNA fragments. A large fraction of the numerous rDNA units are heavily methylated, being completely resistant to Hpa II and Hpa I. However, specific sites are constantly available in another fraction of the units and are therefore unmethylated. Although site-specific methylation of eukaryotic DNA is considered to play a key role in gene regulation (13,26,31), there is so far very little information concerning higher plant genes. It has been known for a long time that higher plant DNA has a much higher 5-methylcytosine level than animal DNA (29), and plant DNA is usually considered to be heavily methylated (18,19 (Fig. 1). This fragment spans the 3' moiety of the 18S r RNA sequences, the internal transcribed spacer, and the 5' part of the 25S rRNA sequence (11). Bacteriophage A RA 2 results from the cloning of Eco RI partial digests of radish DNA into A gt WES/A B vector (22).

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(3,11,14,15,19,21In this paper, we characterize radish 12 and 1.7 S proteins and compare them to their rapeseed equivalents. SDS-polyacrylamide gel patterns and amino acid compositions are reported. In addition we have analyzed each family using two different two-dimensional gel electrophoresis systems. Combining isoelectrofocusing in the first dimension and SDS in the second, we demonstrated that the 12 S family is much more complex than previously assumed. Finally, using a second system in which the first dimension is run in nonreducing conditions whereas the second is in the presence of a reducing agent, we obtained information concerning the associations between the different polypeptides. MATERIALS AND METHODS Extraction and Purification of Storage Proteins. Radish seeds (cv Rond rose a bout blanc, Vilmorin) and rapeseeds (cv JetNeuf) were ground in liquid N2 and extracted as described previously (5). The extract was fractionated into aliquots which were stored frozen at -80°C until use. The 12 and 1.7 S storage protein particles were purified by gel filtration through a Sepharose 6B column (90 x 1 cm) equilibrated in 10 mm sodium phosphate, 0.5 M NaCl, pH 7.5. Fractions were dialyzed against distilled H20, and freeze-dried. Each fraction was then analyzed on a SDS-polyacrylamide gel and those containing the storage proteins were identified. In subsequent preparations, the elution pattern turned out to be very reproducible and the fractions containing the storage proteins were immediately pooled, dialyzed, and freeze-dried. The protein content in each fraction was determined using the Lowry method (20).Polyacrybmide Gel Electrophoresis. To separate proteins in one dimension, the SDS-polyacryLamide gel system (18) was employed, using either 12.5 or 17% slab gels. Gels were stained with amido black or Coomassie blue R-250. Amido black-stained gels were routinely destained and further processed with the silver nitrate staining procedure (24). The isoelectrofocusing cylindrical gels and the second dimension were run as previously described (23, 25) using a pH range 3.5 to 10. The second twodimensional gel system was essentially as already published (22). The first dimension was run on a 1-mm thick slab containing 12.5% acrylamide gel in the absence of2-mercaptoethanol. Each sample was run in duplicate. One strip was stained with Coomassie blue and the other was incubated in sample buffer containing 5% 2-mercaptoethanol to reduce the protein subunits and was placed on the stacking gel of a second dimension 1