Legumin Svnthesis in Developing Cotyledons of Vicia.Faba L.1 Received for Publication March 9,1971

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 purified 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. (16). The intact prepara- TUBE NUMBER tions each gave a single band, but on treatment with urea four bands were obtained from legumin and two from vicilin. As FIG. 1. A typical separation of legumin (I) and Nvicilin (II) by reported (2), dissociation of legumin with SDS gave three centrifugation through a sucrose gradient. Partially purified legu- subunit species, however, our estimates indicate a molecular min and vicilin were applied to a sucrose gradient 5 to 20%) in weight between 18,000 and 20,000 for the smallest subunit. 10 mM tris, pH 7.8, (M----M) or in 10 mm tris, pH 7.8, con- and Vicilin. The fc Amino Acid Composition of Legumin taining 0.5 M NaCl (0 O), and centrifuged amino acid of and vicilin were quite an International SB283 rotor. Fractiions (0.4 ml) compositions legumin 40,000 rpm in similar but the content was differ- were collected and assayed for protein. From the gradient con- (Table I), lysine significantly taining tris-NaCl, fractions 14 to 17, and 22 to 24 were pooled, ent. The analyses are similar to those of Jackson et al. (16), concentrated, and examined by acrylamide gel electrophoresis. and perhaps any differences may be attributed to the use of PlantPtsV41LEGUMINPhysiol. Vol. 48, 1971 SYNTHESIS IN VICIA FABA 421 different cultivars. In contrast with Bailey and Boulter (2), diffusion technique (24). Purified legumin reacted rapidly with cysteine was not present in our legumin preparations. the antiserum to give a strong precipitin band. On prolonged diffusion a second, weaker precipitin band appeared, which PRODUCTION AND TESTING OF ANTILEGUMIN gave a reaction of identity with purified vicilin. This showed AND ANIVICILIN that the purified legumin contained a small amount of vicilin, which was not detected by electrophoresis on acrylamide gels. The legumin preparation was used to produce antibodies in Controls, consisting of protein extracted from mature roots or rabbits. The serum was tested by the Ouchterlony immuno- leaves of V. faba, gave no precipitin bands in diffusions with Downloaded from https://academic.oup.com/plphys/article/48/4/419/6091415 by guest on 24 September 2021

FIG. 2. Behavior of legumin and vicilin in the Ouchterlony faba 4; fresh seeds of P. sativum 5; radicles from V. faba 6; immunodiffusion test. A and B: Immunological identity of purified antiserum 7. Plant tissues were extracted with 50 mm tris, pH 7.8, legumin and vicilin with globulins present in radicles and dried containing 0.2 M NaCl. C: The reaction of antilegumin after ab- seeds of V. faba and in fresh seeds of P. sativum. Gels made of sorption of the serum with vicilin. Purified legumin + vicilin 2, (A) agar-saline (B) agarose-buffer. Purified legumin + vicilin 1; 7; legumin 1, 3; vicilin 6, 8; antiserum absorbed with vicilin 4; purified legumin 2; fresh seeds of V. faba 3; dried seeds of V. antiserum 5. 422 MILLERD, SIMON, AND STERN Plant Physiol. Vol. 48, 1971 the antiserum, showing that adult roots and leaves are free of antibodies in the rabbit serum was obtained by treating the legumin and vicilin. serum with an excess of vicilin. Figure 2C clearly shows that It has been suggested that legumin and vicilin are closely after absorption of the serum it no longer reacts with vicilin related, have sequences of amino acids in common (16), and but reacts strongly with legumin. that immunologically they cross react reciprocally (19). There When legumin or vicilin were tested on the usual Ouchter- are two possible explanations for this cross-reactivity. The first lony plates (agar in saline), in addition to the specific precipi- is that it could arise through incomplete separation of vicilin tin bands, halos appeared around the wells containing the pro- from legumin and the consequent cross-contamination of the tein (Fig. 2A). Indeed, with extracts of cotyledons, nonspecific preparations used in injecting the animals to prepare the anti- precipitin bands formed with control, zero-bleed, sera of rab- sera. The second alternative is that legumin and vicilin really bit and sheep. These artifacts are avoided by using plates pre- do share antigenic determinants and would therefore always pared in tris, EDTA, boric acid buffer pH 9.2 (Aronsson and show some cross-reactivity even with antisera prepared against Gronwal, quoted by Bodman [4]), and best resolution is at- absolutely pure antigens. The double bands (Fig. 2B) that do tained using agarose prepared in this buffer (Fig. 2B). not spur between legumin (2) and legumin plus vicilin (1) In these investigations, globulins were isolated from freshly- Downloaded from https://academic.oup.com/plphys/article/48/4/419/6091415 by guest on 24 September 2021 favor the first explanation. This is also the evidence of Figure harvested material. It seems possible that some breakdown of 1 (4) in Kloz and Turkova (19) and is in agreement with the the globulins could occur during seed maturation. However, data of Graham and Gunning (14) with fluorescein isothio- legumin and vicilin extracted from desiccated seeds were im- cyanate conjugates of antilegumin and antivicilin. munologically identical (Fig. 2B) with those present in fresh Additional evidence for the presence of specific antilegumin seeds. In addition, the globulins of V. faba were immunologically identical with those of P. sativum (Fig. 2B). Legumin A- and vicilin were not restricted to the cotyledons but were also present in the radicle (Fig. 2B) as was indicated by Ghetie and Buzila (13). Klein and Pollock (18), using radicles from 1.2- s~~~~~~ 0 Phaseolus lunatus L., have reported electron dense bodies which they considered to be globulins. We suggest that globu- z lins are present in tissues of the embryo but are absent from o 1.0 h 0 corresponding mature organs. LU H 0.8 I. 0 0 PARAMETERS OF COTYLEDON DEVELOPMENT IU 00.6 Size. As a measure of development, the length of the long H axis of the cotyledon was a reliable criterion. During the (2 growth period studied, the length of the cotyledon correlated LLJLU C: 0.4 II, 0 well with the fresh weight, particularly in the early stages of development (Fig. 3). The maximum cotyledon length ob- LI) served was 32 mm. This measurement was found easier to L. 0.21 obtain, and indeed more reliable under the growing conditions used, than the classification "days from flowering." For com- 0 parison with other reports, it was observed that a cotyledon 0 4 8 12 16 20 24 l I28I was 19 to 21 mm in length about 32 days from opening of the flower and that a cotyledon approximately doubled in LENGTH OF COTYLEDON (mm) length in 7 days. FIG. 3. Relationship of length and fresh weight of cotyledons. Cell Number. In a developing plant organ there are two phases of growth, an initial one of cell division and a later 1X107 I I I X one of cell expansion. During cotyledon growth, cell number increased rapidly and essentially the full complement of cells, approximately 2 million, was reached when the cotyledon was about 9 to 10 mm long (Fig. 4). After this, only occasional mi- 0 0 totic figures could be seen. Chlorophyll Content. As a measure of synthesis not directly ~~~~~~~~~~~~~~ associated with synthesis of storage proteins, the chlorophyll zJ~~1.106 _ ~ ~~09~~~~ content of the cotyledons was determined. The chlorophyll content increased steadily with development (Fig. 5); there 0C: 0 was no marked change in the rate of chlorophyll accumula- LU tion.

CORRELATION OF COTYLEDON DEVELOPMENT WITH LEGUMIN CONTENT C-l The protein and legumin contents of cotyledons at different stages of development were determined. Cotyledons at all stages of development were collected and, with NaCl-buffer, protein was extracted from pooled samples of each size. The 1X104 . _ I 10 12 14 protein and legumin contents of the extracts were then de- 2 4 6 8 termined. LENGTH OF COTYLEDON em m)~ In order to use the antiserum for reliable measurements of FIG. 4. Relationship of length and number of cells per cotyledon. legumin content, it was necessary to define conditions under Plant Physiol. Vol. 48, 1971 LEGUMIN SYNTHESIS IN VICIA FABA 423

z 0 o LU -j 101

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1L 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 LENGTH OF COTYLEDON (mm) FIG. 5. Change in content of chlorophyll a + b with development of cotyledon. which a specific legumin-antilegumin reaction could be measured exclusively. Fortunately, the antilegumin titer was much greater than the antivicilin titer. At appropriate dilutions in the microcomplement fixation assay, the serum reacted only C with legumin (Fig. 6). To determine whether vicilin would cs inhibit the legumin reaction increasing amounts of vicilin were CV) added to the reaction mixture. No inhibition was observed even when vicilin was present in 15-fold excess over the concentration of legumin. The data in Table II almost certainly z co reflect the developmental changes in legumin content and are 0cC unaffected by the presence of vicilin in the extracts. CID With growth, the amount of extractable protein per cotyledon increased rapidly (Table II). Below 7 mm in length, a A cotyledon contained less than 1 ,ug of legumin (Table II). After attaining 10 mm in length there was a marked increase in 0 legumin content. This is more clearly demonstrated by plotting *50 1*0 (Fig. 7) legumin as a percentage of the extractable protein. pg ANTIGEN To determine whether these measured increases in legumin titer were due to a single species of molecule or to crossre- FIG. 6. Reaction of antilegumin with legumin and vicilin. Mi- acting antigens, extracts of cotyledons from different develop- crocomplement fixation reaction of antilegumin at a dilution of 1:6,000 with different amounts of legumin (O-O) and vicilin mental stages were each analyzed for legumin versus protein (@----e). In the microcomplement fixation assay, the final content. The results are shown in Figure 8. Clearly, although step measures decrease in the lysis of red cells and hence a re- there are extreme differences in the legumin to protein ratio duced absorption due to hemoglobin (413 nm). in extracts from different stages, the peaks formed in the microcomplement assay have similar heights regardless of the value of that ratio. Such a family of plots has been considered shift which occurs in the pattern of protein synthesis during to be satisfactory proof that a single molecular species is being cotyledon development as was shown in Figure 7. measured (20). Figure 8 illustrates incidentally the dramatic Microcomplement fixation could not be used to assay vicilin 424 MILLERD, SIMON, AND STERN Plant Physiol. Vol. 48, 1971 in these extracts. At serum dilutions necessary for vicilin as- Table II. Content of Legumin and Total Extractable Protein says, there was interference from legumin. during Development of Cotyledon Samples of each size of cotyledon were exhaustively extracted DISCUSSION with 50 mm tris, pH 7.8, containing 0.2 M NaCl. After centrifu- The results reported here contribute further to the details gation, the protein and legumin contents of the extracts were of protein synthesis in the developing bean cotyledon and lend determined. additional emphasis to the usefulness of the bean cotyledon as a system for the study of cell differentiation. A strong effort Length of Cotyledon Extractable Protein Legumin was made to obtain precise determinations of the legumin con- mint mg/cotyledon j1g/cotyledon tent of the cotyledons with special emphasis being put on the 3 0.03 4 0.10 5 0.22 0.2 27k 6 0.54 0.6 Downloaded from https://academic.oup.com/plphys/article/48/4/419/6091415 by guest on 24 September 2021

26 - 7 0.92 1.2 / 8 1.0 1.2 9 1.4 2.2 10 1.7 20t 11 2.75 18.7 12 3.2 38.4 13 3.5 84 z L 14 5.6 140 m 0 15 280 0 8.0 i5~5- 0 16 5.6 263 15e 17 7.7 716 -J 18 10.0 700 ccc- atU 19 8.4 840 c- 20 14.7 1.91 X 103 wx 21 15.4 22 31.1 23 30.2 6.34 X 10' ! 10 z 24 43.7 6.99 X 10' 0 25 39.2 10.58 X 10' w 26 53.5 -AJ 27 68.3 28 51.2 8.8 X 103

5 F- L specificity of measurements. The checks which we have made on the microcomplement fixation assay give confidence that the developmental data presented do in fact represent the course of legumin accumulation during cotyledon growth. What stands out is the sharp increase in the rate of legumin 10 15 20 25 30 accumulation when the cotyledons were about 10 mm in length. At this stage cell division was essentially complete, LENGTH OF COTYLEDON (mm) and subsequent growth was by cell expansion. Increased legu- Flo. 7. Change in legumin content of cotyledon with develop min accumulation was not paralleled by a general increase in ment. protein. As shown in Figure 7, the ratio of legumin to other

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pg PROTEIN /ml EXTRACT FIG. 8. Microcomplement fixation of antilegumin with different amounts of cotyledon extract prepared from cotyledons at different stages of development. The size of cotyledon for each set of analyses is shown in the figure. The significant result in this experiment is the similarity in maxima reached for each stage regardless of the ratio of antigen to protein. Plant Physiol. Vol. 48, 1971 LEGUMIN SYNTHESIS IN VICIA FABA 425

increased with of The nature of the 14. GRAHAM, T. A. AND B. E. S. GUN G. 1970. Localization of legumin and vi- proteins length cotyledon. cilin in bean cotyledon cells using fluorescent antibodies. Nature 228: 81-82. low level of legumin in younger cotyledons is unclear. There 15. GRANT, D. R. AND J. M. LAWRENCE. 1964. Effects of sodium dodecyl sulfate may be a slow rate of synthesis in all cells or there may be a and other dissociating reagents on the globulin of peas. Arch. Biochem. high rate of synthesis in a few cells. The data of Graham and Biophys. 108: 552-561. 16. JACKSON, P., D. BOUJLTER, AND D. A. THuaRN. 1969. A comparison of some Gunning (14) would favor the second possibility, but their properties of vicilin and legumin isolated from seeds of Pisum sativum, report, which is based on microscope studies of immuno- Vicia faba and Cicer arientinum. New Phytol. 68: 25-33. fluorescence, lacks the quantitation for a closer comparison 17. JOHNSON, P. AND E. G. RICHARDS. 1962. The study of legumin by depolariza- with the results obtained in these studies. tion of fluorescence and other physicochemical methods. Arch. Biochem. Biophys. 97: 260-276. Acknowledgments-The authors are grateful to Dr. R. Doolittle, University of 18. KLEIN, S. AND B. M. POLLOCK. 1968. Cell fine structure of developing lima California, San Diego, for amino acid analyses; to Miss Ann Roman, University bean seeds related to seed desiccation. Amer. J. Bot. 55: 658-672. of California, San Diego, for determination of cell numbers, to Dr. W. Dudman, 19. KLoz, J. AND TURKOVA. 1963. Legumin, vicilin and proteins similar to them CSIRO, Canberra, for variations of the Ouchterlony immunodiffusion technique, in the seeds of some species of the Viciaceae family (a comparative serologi- and to Dr. J. Jacobsen, CSIRO, Canberra, for studies on dissociation of legumin cal study). Biol. Plant. (Praha) 5: 29-40. by SDS. 20. LEVINE, L. AND H. VAN VUNAKIS. 1967. Micro complement fixation. In: C. H. Downloaded from https://academic.oup.com/plphys/article/48/4/419/6091415 by guest on 24 September 2021 W. Hirs, ed., Methods in Enzymology, Vol. XI. Academic Press, New York. pp. 928-936. LITERATURE CITED 21. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Pro- tein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265- 1. ARNON, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase 275. in Beta vulgaris. Plant Physiol. 24: 1-15. 22. MOORE, S. AND W. H. STEIN. 1963. Chromatographic determination of amino AND The structure of a 2. BAILEY, C. J. D. BOULTER. 1970. legumin, storage pro- acids by the use of automatic recording tein seed. Eur. J. Biochem. 17: 460-466. equipment. In: S. P. Colowick and of broad bean (Vicia faba) N. 0. eds., Methods in Enzymology, Vol. Academic Press, New AND D. 1970. A slice for Kaplan, VI. 3. BAILEY, C. J., A. COBB, BOULTER. cotyledon system York. pp. 819-831. the electron autoradiographic study of the synthesis and intracellular trans- 23. G. of the seed of Vicia Planta 95: 103-118. MoRRss, F. I., D. A. THURMAN, AND D. BOUmTER. 1970. The extraction and port storage protein faba. chemical composition of aleurone grains (protein bodies) isolated from 4. BODMAN, J. 1960. Agar gel, starch block, starch gel and sponge rubber electro- seeds phoresis. In: I. Smith, ed., Chromatographic and Electrophoretic Tech- of Vicia faba. Phytochemistry 9: 1707-1714. Vol. London. 96. 24. OUCHTERLONY, 0. 1949. Antigen-antibody reactions in gel. Acta Path. Micro- niques. Zone Electrophoresis, II. Heinemann, p. biol. Scand. 26: 507-515. 5. BONNER, J. 1965. The Molecular Biology of Development. Oxford University Press. London. 25. PAYNE, P. I. AND D. BOULTER. 1969. Free and membrane bound ribosomes of 6. BONNER, J. 1965. The template activity of chromatin. J. Cell. Comp. Physiol. the cotyledons of Vicia faba (L.) I. Seed development. Planta 84: 263-271. Supp. 66, 77-90. 26. RIJVEN, A. H. G. C. AND I. F. WARDLAW. 1966. A method for the determination 7. BONNER, J., M. E. DAscMUs, D. FAmBOROUGH, R. C. HUANG, and D. Y. H. of cell number in plant tissues. Exp. Cell Res. 41: 324-328. TUAN. 1968. The biology of isolated chromatin. Science 159: 47-56. 27. SHuJrov, A. D. AND I. A. VAINTRAuB. 1966. Chromatographic isolation and 8. BONNER, J., R. C. HUANG, AND R. V. GILDEN. 1963. Chromosomally directed some properties of the legumin and vicilin of vetch. Biochemistry (U.S.S.R.) protein synthesis. Proc. Nat. Acad. Sci. U.S.A. 50: 893-900. 31: 634-641. 9. BOULTER, D. 1965. Protein biosynthesis in higher plants. In: J. B. Pridham and 28. VAINTRAUB, I. A., A. D. SHUTOV, AND V. G. KLImENxo. 1962. The globulins of T. Swain, eds., Biosynthetic Pathways in Higher Plants. Academic Press, vetch seeds. Biochemistry (U.S.S.R.) 27: 293-305. London. pp. 101-115. 29. VARNER, J. E. AND G. SCaDLOVsKY. 1963. Intracellular distribution of proteins 10. BRIARTY, L. G., D. A. COULT, AND D. BOULTER. 1969. Protein bodies of devel- pea cotyledons. Plant Physiol. 38: 139-144. oping seeds of Vicia faba. J. Exp. Bot. 20: 358-372. 30. WASSERMAN, E. AND L. LEVINE. 1961. Quantitative micro-complement fixation 11. DANIELssoN, C. E. 1949. Seed globulins of the Gramineae and Leguminosae. and its use in the study of antigenic structure by specific antigen-antibody I. Seed globulins of the most common species of the Gramineae and their inhibition. J. Immunol. 87: 290-295. differentiation in the seed. Biochem. J. 44: 387400. 31. WEBER, K. AND M. OssoRN. 1969. The reliability of molecular weight determi- 12. DANIELBSON, C. E. 1950. An electrophoretic investigation of vicilin and legu- nations by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. mi from seeds of peas. Acta Chem. Scand. 4: 762-771. 244: 4406-4412. 13. GHETE, V. AND L. BuzILL. 1962. Cercet'ari Imunochemice asupra germinatiei 32. WHEELER, C. T. AND D. BOULTER. 1967. Nucleic acids of developing seeds of la bob (Vicia faba). Stud. Cercetbioch. 5: 541-550. Vicia faba L. J. Exp. Bot. 18: 229-240.