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Plant Physiol. (1971) 48, 419-425 Legumin Svnthesis in Developing Cotyledons of Vicia.faba L.1 Received for publication March 9,1971 ADELE MILLERD,2 M. SIMON, AND H. STERN Department of Biology, University of California, San Diego, La Jolla, California, 92037 ABSTRACT discarded. The seed coats were removed and the plant axes, which were well defined, were removed by dissection. Downloaded from https://academic.oup.com/plphys/article/48/4/419/6091415 by guest on 24 September 2021 The synthesis of legumin in developing cotyledons of Vicia Isolation of Legumin and Vicilin. Cotyledons were blended faba L. has been examined as a potential system for approach- with 2 volumes (w/v) 50 mm tris buffer, pH 7.8, containing ing the problem of differential gene expression. The pattern 0.2 M NaCl (NaCl-buffer), and the resulting brei was stirred of legumin synthesis was determined during the growth of the at 4 C for 1 hr. The brei was filtered through Miracloth, and cotyledon by microcomplement fixation which provided a the filtrate was centrifuged at 4000g for 10 min. The super- sensitive and specific assay for legumin in the presence of natant was brought to 40% saturation with (NH4)S04, and the vicilin. Legumin was detected even in young cotyledons. How- precipitate was discarded. The supernatant was brought to ever, when the cotyledons were about 10 millimeters long, and 70% saturation with (NH,)1S04. The resulting precipitate was cell division was essentially complete, there was a sharp in- dissolved in 10 mm tris buffer, pH 7.8, and refractionated crease in the rate of legumin accumulation. with (NH,)-SO,. The material precipitating between 42.5 and 65% saturation was recovered, dissolved in 10 mm tris buffer, pH 7.8, and dialyzed at 4 C against 10 mm tris, pH 7.8, until incipient precipitation. This dialyzed material was then puri- fied by centrifugation through a sucrose gradient (see "Re- sults"). Analytical Gel Electrophoresis. Legumin and vicilin were Developing seeds of Leguminosae offer an attractive system examined on 7.5% acrylamide gels at pH 8.9 in tris buffer. for studying the problem of differential gene expression. These The proteins were dissociated with 8 M urea and run on 7.5% seeds accumulate large amounts of globulins which consist of acrylamide gels in the presence of 8 M urea at pH 8.9 (tris), two high molecular weight proteins, legumin and vicilin, as de- pH 6.5 (cacodylate) and pH 4.4 (acetate). The gels were run fined by analytical centrifugation (11, 12). Varner and Schid- for 5 hr at 150 v. The presence or absence of smaller lovsky (29) showed globulins to be localized in protein bodies molecular weight proteins was checked by running gels for within pea cotyledons. Jackson et al. (16) analyzed legumin shorter periods using bromophenol blue as tracking dye. Legu- and vicilin from Vicia faba L. by fingerprinting tryptic digests min was dissociated (31) with SDS,' and molecular weights and identifying the N-terminal amino acids. The subunits of of the subunits was estimated (31). legumin have been studied by Bailey and Boulter (2). Morris Amino Acid Analyses. Duplicate samples were hydrolyzed et al. (23) isolated the protein bodies of V. faba and deter- in 6 N HCI at 110 C for 24 hr, and the amino acid composi- mined their chemical composition. Graham and Gunning (14) tions of legumin and vicilin were determined according to localized the two proteins in bean cotyledon cells by means of Moore and Stein (22). For more accurate assays of valine and fluorescent antibodies. isoleucine, 48-hr acid hydrolysis was used. A major attempt to use developing seeds of Leguminosae Production of Antibodies. About 1 to 2 mg of purified legu- for studies of differential gene expression was first made by min was mixed with Freund's adjuvant and injected into the Bonner et al. (5-8). They used immature seeds of Pisum sati- toe pads of a rabbit. Three weeks later an additional 1 mg of vum for analyzing the regulation of transcription and transla- legumin was given intravenously and 1 week after that serum tion of chromatin preparations from cotyledon cells. More of high titer was collected. recently, Boulter and associates (2, 3, 9, 10, 25, 32) have Extraction of Protein from Cotyledons. Cotyledons were initiated extensive studies of cotyledon development in V. homogenized in NaCl-buffer, the brei was centrifuged faba. In this paper we wish to report our analyses of legumin (27,000g, 20 min), and the supernatant was removed. The synthesis in relation to the growth of the cotyledon of V. faba. residue was re-extracted, and the procedure was repeated until no further protein was obtained. The protein present in the MATERIAIS AND METHODS combined supernatants, the extractable protein of Table II, accounts for approximately 95% of the total protein of the Plant Material. Seeds of Vicia faba L. (cultivar unknown) cell, since a further 5% was extracted from the residue by were grown in soil in the greenhouse or garden. When pods treatment with 0.1 N NaOH. Protein was assayed according were harvested, any abnormally shaped material was excluded. to Lowry et al. (21) using bovine serum albumin as standard. In a pod there were usually three or four seeds of equal size Estimation of Legumin using Antilegumin. The estimation which were used and one or two smaller seeds which were of legumin was carried out by the microcomplement fixation method of Wasserman and Levine (30). Until the cotyledons 1Research was supported by a grant from the National Science were in an advanced developmental stage, extracts from them Foundation (GB-5173X) and by a grant from the National Insti- showed negligible complement-binding activity. The antiserum tutes of Health (USPHS HD 03015). "Permanent address: Division of Plant Industry, CSIRO, Can- bera, A.C.T. 2601, Australia. 8Abbreviation: SDS: sodium dodecyl sulfate. 419 420 MILLERD, SIMON, AND STERN Plant Physiol. Vol. 48, 1971 contained antibodies against both legumin and vicilin but was Table I. Amino Acid Compositioni of Legumin and Vicilin used at a dilution at which the antivicilin did not interfere. The amino acid compositions were determined after acid hy- Cell Number. The number of cells per cotyledon was de- drolysis and are expressed as a percentage of the total amino acids termined as described by Rijven and Wardlaw (26) except that recovered. Tryptophan and amide-N were not determined. pectinase, instead of cellulase, was used to disperse the cells. were extracted into Amino Estimation of Chlorophyll. Pigments AminoAminoAcidsAcids ~ CompositionAcidViliLegumin Vicili 80% acetone and the concentration of chlorophyll a + b was determined as described by Arnon (1). mole percent Lysine 4.2 6.5 RESULTS Histidine 2.4 1.8 Arginine 8.0 6.4 ISOLATION AND CHARACTERISATION OF LEGUMIN Aspartic Acid 12.3 12.2 AND VICILIN 3.7 3.8 Threonine Downloaded from https://academic.oup.com/plphys/article/48/4/419/6091415 by guest on 24 September 2021 Isolation. Globulins were isolated from cotyledons of Serine 7.4 7.5 freshly harvested seeds. Seeds selected were in advanced stages Glutamic Acid 19.9 17.4 of development but prior to the onset of dehydration. Globu- Proline 5.4 4.6 lins were extracted and partially purified by fractional precipi- Glycine 7.7 6.5 tation with ammonium sulfate. Alanine 6.3 6.0 Legumin and vicilin were then separated on a sucrose gradi- Cystine/2 0.0 0.4 ent. Separation of the globulins was considerably sharpened Valine 5.1 5.6 by the presence of NaCl in the gradient (Fig. 1). Protein was Methionine 0.3 0.7 regions of the gradient (I, II, III, Fig. 1). Isoleucine 4.3 5.1 distributed in three Leucine 8.5 8.9 Several gradients were developed and the fractions directly 2.7 were and concentrated by perevapora- Tyrosine 2.1 under each peak pooled Phenylalanine 3.2 4.0 110 tion during dialysis against 10 mM tris, pH 7.8, containing 0.15 M NaCl. The three fractions were examined by disc ioo electrophoresis on acrylamide gels. Present in the light region (III, Fig. 1) of the gradient were proteins of low molecular weight, presumably of cytoplasmic 90 origin. In the dense region of the gradient (I, Fig. 1), a single protein species, corresponding to legumin, was detected. The 80 intermediate fraction (II, Fig. 1) corresponding to vicilin, showed two bands on acrylamide gel and a trace of low molecular weight protein. It is possible that both the strong 70 bands of protein corresponded to vicilin, perhaps monomer 7~- and dimer, as dissociation of fraction II with urea gave only z 60 two subunits. ULJ Considerable difficulty has been encountered in separating 0 completely legumin from vicilin by ammonium sulfate precipi- 50 tation (11, 19, 28). Chromatography on diethylaminoethyl cellulose has also been used (15, 27). In our hands, this yielded 40 legumin contaminated with small molecular weight proteins. Gel filtration has been used (16) and also repeated isoelectric precipitation (2, 16). When our preparations obtained by su- 30 crose density-gradient centrifugation were checked with anti- > ts sera using the Ouchterlony immunodiffusion technique (24), 20 it was shown that legumin contained a trace of vicilin, but vicilin could be prepared free from legumin. ' Purified legumin and vicilin were stored frozen in buffer 10 containing 0.15 M NaCl. In the absence of NaCl, on freezing and thawing, legumin and vicilin precipitated and would not 28 redissolve even if NaCl were added. Analyses of the prepara- 1I tions by acrylamide gel electrophoresis yielded results similar 20% SUCROSE 5% SUCROSE to those reported by Jackson et al.

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