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Formation of Protein Bodies in Ripening Seeds of Vicia faba L.
Biochem. Physiol. Pflanzen 173, 167-180 (1978)
Formation of Protein Bodies in Ripening Seeds of Vicia faba L. D. NEUMANN and E. WEBER Institut fiir Biochemie der Pflanzen, DDR - 402 Halle (Saale) und Zentralinstitut fiir Genetik und Kulturpflanzenforschung, DDR - 4325 Gatersleben, der Akademie der Wissenschaften der DDR Key Term Index: aleurone grains, electron microscopy, development; Vicia (aba.
Summary The development of aleurone grains in ripening Vicia (aba seeds is investigated by electron microscopy and by biochemical methods. The cotyledonary cells persist in an embryonal stage during three weeks after anthesis, later they show a typical picture with stacks and strands of rough ER, stained vesicles in connection with dictyosomes and a large central vacuole. This vacuole disappears, but in the stage of reserve protein production a new vacuolar system - the aleurone grains - si formed by evagination and swelling of ER membranes. The role of the dictyosomes for the production of storage protein is discussed.
Introduction
Endosperm and cotyledons of many plant species accwnulate storage material of high uniformity in large quantities. It can be mobilized during germination and then used for the growth of the embryo in early stages of development. The origin of the storage compartment is unclear or not yet investigated in most cases. For the storage of proteins in protein bodies three compartments are discussed: plastids, vacuoles and cisternae of the endoplasmic reticulum (ER). MORTON and coworkers (1963, 1964) described protein bodies of wheat endosperm originating from plastids, since two envelope membranes could be dicriminated; however, this observation was disapproved by KHOO and WOLF (197Q). In some other species protein bodies are surrounded by only a single membrane, "very similar" to the tonoplast of Gossypium (YATSU 1965; ENGLEMAN 1966), Arachis (DIECKERT and DIECKERT 1976), Hordeum (ORY and HENNINGSEN 1969), Vitia (BRIARTY et al. 1970), Sinapis (REST and VAUGHAN 1972), Phaseolus (HARRIS and CHRISPEELS 1975). In some cases a central vacuole is filled successively with osmiophilic material identified as protein (DIECKERT and DIECKERT 1976; REST and VAUGHAN 1972). In contrast, protein bodies of other species are formed by separation from ER cisternae (KHOO and WOLF 1970). With the exception of the reserve proteins in wheat endosperm it seems obvious that protein bodies are surrounded by a single membrane only. The origin of this membrane is not clear in most cases. At present it is unknown whether the possibilities mentioned here can exist in the same tissue or in the same species.' In the present paper the development of protein bodies in Vicia faba is studied by means of electron microscopy and biochemical methods.
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Material and Methods Plant Material Vicia (aba L., var. minor, cv. "Dornburger Ackerbohne" and cv. "Fribo" were used as materials for these experiments. During spring and summer 1976 and 1977 fully opened flowers of the 2nd, 3rd and 4th node of green house plants were labeled. After 18 days plant material was successively harvested daily until the 50th day after flowering. For analysis pods of average size containing 4 seeds were selected. Growth Oharacters
Length of seeds and cotyledons (long axis) as well as fresh and dry weights were chosen as basic morphological growth characters. Nitrogen Determinations
Whole cotyledons of early developmental stages, which were dried at 105°C, or aliquots of about 30 mg were analysed by a modified Kjeldahl method after FLECK and MUNRO (1965). 3H-Leucine Incorporation into Globulins
Freshly harvested seeds were dehulled and the testa, radicle, and plumula were dissected. Cotyledons were incubated according to PUCHEL (in preparation) for 2 h in 1 ml incubation medium of MILLERD et al. (1975) containing 50 I'ci 3H-Leu (58 Ci/mMol). The incubation flasks were shaken in the dark at 25°C and the reaction stopped by addition of ice water. Samples were stored at -20°C until used.
of Labeled Globulins Two cotyledons were homogenized with a BUhler homogenizer for 5 min with 10 ml 0.1 M phosphate buffer, pH 7.0, containing 0.5 M sodium chloride and stirred for 1 h in the same medium. Cell debris were removed by a 3000 . g centrifugation. All procedures were carried out at 4°C. The globulins legumin and viciIin were immunoprecipitated from aliquots of the extracts using an excess of monospecific antisera for legumin and vicilin, which were produced as described elsewhere (SCHOLZ et al. 1974). Extraction and Determination
Electron Microscopy
Small pieces of cotyledons were fixed with 3 % glutaraldehyde in 0.05 M cacodylate buffer pH 7.4 (2 h) and 1 % OsO, in the same buffer (1 h). For washing 0.1 mol cacodylate buffer pH 7.4 was used. Following block staining (2 % uranyl acetate in 75 % acetone, 2 h) the specimens were embedded in Araldit. Ultrathin sections cut with glass knives were stained with lead acetate and viewed in a transmission electron microscope (SEM 3/2, Werk fur Fernsehelektronik Berlin). The pronase digestions (0.01 % pronase in phosphate buffer pH 7.4, 1 h) were carried out on ultrathin sections after bleaching in 10% H2 0 2 (10 min).
Results
The increase of dry weight, fresh weight and total N of seeds and cotyledons show a typical sigmoid curve. After a lag phase of 35 days the total N content of the cotyledons increases linearly between 39 and 43 days. The rate of increase is reduced after 50 days (Fig. 1) Fresh weight and dry weights of seeds and cotyledons increase similarly (Fig. 2). The fresh weight increases mor,e in comparison with the dry weight between 28 and 32 days, but after 43 days the rate of increase of both growth parameters decreases similar to the N content. The curves seem to approach a stationary phase,
169
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Fig. 1. Total N content of the developing cotyledons of Vicia faba. Modified Kjeldahl method after FLECK and MUNRO (1965); abscissa: days after anthesis; ordinate: mg N/2 cotyledons. Fig. 2. Fresh weight and dry weight of cotyledons and seeds of Vicia faba. Absissa: days after anthesis; ordinate: mg fresh weight of seeds (-----), mg fresh weight/2 cotyledons ( - - ) , mg dry weight/2 cotyledons (-. - . -).
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Fig. 3. Increase in the length of developing seeds and cotyledons of Vicia faba. Abscissa: days after anthesis; ordinate: mm; seeds (---), cotyledons (- - - - -).
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which was already demonstrated by MANTEUFFEL et al. (1976) in a previous paper. The parallel course' of fT-esh weight and dry weight in later stages of development is an expression of desiccation of the ripening seeds. These changes a~e macroscopically visible in the development of the length of seeds and cotyledons (Fig. 3). They show a constant increase in length between 23 and 47 days after anthesis. After start of the desiccation of the seeds the differences become smaller, and after 48 days length of seeds and cotyledons are approximately equal. In this stage of development the N content is also approximately constant (about 12 mg N/cotyledon, that means about. 33 % ctude protein/dry weight). The seeds have reached their final length and loose their green colour. The incorporation rates of 3H-Ieucine into the globulins vicilin and legumin correspond with the linear phase of the total N-accumulation (compare Fig. 1 and Table 1). The rate of vicilin synthesis is higher than that of legumin during the first 8 days. Later the rate of legumin synthesis increases r,apidly while the rate of viciIin formation seems to decrease (Table 1). The interpretation of these differences need further experimental work, especially by immunocytochemical methods. Tabelle 1. Incorporation of 3H-leucine into storage proteins of Vicia faba during seed development. Liquid scintillation counting after immunoprecipitation with monospecific antisera of the radioactively labelled globulins vicilin and legumin. Numbers are d.p.m./aliquot days after flowering
vicilin
legumin
29 33 37 43 44 48
200 11,100 12,670 8,750 9,760 8,040
170 7,200 8,510 26,280 72,930 47,590
Electron microscopic investigations demonstrate that biochemically well characterized phases in the development of seeds are connected with typical changes in the ultrastructure of the cells. In the following the development of the cotyledons is classified in definite phases by characteristic pictures on the ultrastructural level. This classification is valid o'nly for the investigated Vicia faba val). minor, cv. "Dornburger". Other cultivars (e.g. Vicia faba var. minor, cv. "Fribo") show a similardevelopment, but a temporal shift is observed, e.g. 10 days delay for "Fribo" in comparison with "Dornburger", which is pn,obably due to different cultural conditions during spring and summer.
Phase 1 (0-24 days; Figs. 4, 5, 15A) In the first days after anthesis the metabolically active cotyledonary cells show' a dense cytoplasm and a folded plasmalemma. The cytoplasm contains small vacuoles and a large number of unstained vesicles derived from dictyosomes. The cells are connec-
Figs. 4-14. Formation of aleurone grains in ripening seeds of Vicia taba var. minor, cv. "Dornburger". Fixation: GlutaraldehydjOs04' Fig. 4. 18 days old cotyledons. Note the folded plasmalemma, dictyosomes (d), mitochondria (m), plastids (p), vacuoles (v) and endoplasmic reticulum. X 21,600. Fig. 5. 25 days old cotyledons; nucleus (n); cell wall (w); X 10,800.
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Fig.6. 29 days old cotyledons. Note the increased vacuolization and formation of rough ER. X 21,600. Fig. 7. 31 days old cotyledons. Numerous strange of rough ER, dictyosomes (d) with osmiophilic vesicles and vacuoles with dark stained material (protein-e). X 13,400.
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173
Fig. 8. 31 days old cotyledons after treatment with pronase. The light parts into the vacuole (v) are formed by enzymatic digestion of protein. X 16,000.
ted together by numerous plasmodesmata. Plastids ave very small with only some thylakoid membranes and few small starch grains.
Phase 2 (25-28 days; Figs. 6, 15B) Within few days drastic changes in the ultrastructure occur. The cells leave their embryonal stage, the number of ER membranes increases. The membranes of the endoplasmic reticulum become occupied by a large number of ribosomes. At the age of 28 days after anthesis stacks of rough ER are enriched in the cytoplasm. Products of the metabolic activity of the ER membranes cannot be localized by the electron microscope, however, this must not exclude protein synthesis, since not all proteins form an insoluble network with glutaraldehyde and can be stained by OS04·
Phase 3 (29-32 days; Figs. 7, 8, 15C) The vacuolization proceeds between 29 and 32 days, and in most of the cells only a large central vacuole is visible. Rough ER strands and stacks occupy most of the cytoplasm. Dictyosomes can be observed frequently p~oducing dark stained vesicles. On the vacuolar surface of the tonoplast and also inside the vacuoles osmiophilic ma-
Fig. 9. 35 days old cotyledons. Reserve proteins are excreted in the enlarged volume of the ER. The aleurone grains (a) may be fused together ( - - ) . X 24,000. Fig. 10-11. 35 days old cotyledons. Enlarged ER cisternae and formation of aleurone grains from the ER membranes. X 24,00; X 16,000.
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Fig. 12. Dictyosomes in a 36 days old cotyledon. The dictyosomes (d) forms osmiophilic vesicles. A participation of these organelles on the production of aleurone grains cannot be seen in the electron microscope. X 32,400. Fig. 13. 40 days old cotyledons. Most of the rough ER disappears and the cell is occupied by large irregular shaped aleurone grains (a). x7,200.
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Fig. 14. 44 days old cotyledons. The proportion of rough ER membranes is decreased; ER membranes divide (-) into spherosomes (s) situated around the plastids (p). X 24,000.
teJ;ial was detected. This mate:r:ial was identified as protein: Treatment of ultrathin sections with pronase removes the osmiophilic material (Fig. 8).
Phase 4 (33 -38 days; Figs. 9 -12, 15 D) Between 33 and 38 days aleurone grains are synthesized in the cotyledonary cells. This process is accompanied by dramatic changes in the ultrastructure of the cells. At first the central vacuole disappears, and within a few days aleurone grains of different size partially occupy the cytoplasmic compartment. Very often aleurone grains are surrounded by stacks of the rough ER. In some cases the formation of aleurone grains from evaginations and segregation of the ER is demonstrated (Figs. 10, 11). In the cytoplasm small vesicles without contrast or dark-stained can be observed connected to dictyosomes (Fig. 12).
Protein bodies of Vicia faba seeds
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Phase 5 (39-43 days; Figs. 13, 15 E) The formation of aleurone grains is completed in this phase. The cells contain numerous large branched aleurone grains and virtually no active dictyosomes. The proportion of rough ER membranes decreases. Phase 6 (older than 44 days; Figs. 14, 15 F) These cells show a smaller amount of rough ER in comparison with cells of the phases 3-5. It seems that smooth ER membranes divide into small vesicles and surround the plastids as spherosomes. Aleurone grains of nearly circular shape (1-2 p,m in diameter) can be observed in the cytoplasm. Fig. 15 shows schematically the formation of aleurone grains in seeds of Vicia {aha. The developing cotyledons consist of embryonic cells without specific differentiation during three weeks after anthesis (Fig. 15 A). At 24 days many stacks and strands of rough ER appear without remarkable protein accumulation at this time (Fig. 15B). Only 4 days later proteinous material is stored in the large central vacuole (Fig. 15 C). In the following time the central vacuole disappear's and a new vacuolar apparatus aleurone grains - is formed by evagination of the rough ER (Fig. 15D). Within a few days only the major quantity of the storage protein is synthesized and stored in all{lUrone grains of various size and irregular shape (Fig. 15 E). Later the rough ER disappears, spherosomes are formed from the smooth ER and the aleurone grains dissociate into small vesicles (Fig. 15F).
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Fig. 15. Schematical hypothesis on the formation of aleurone grains in cotyledons of Vicia faba, A: 0-24d; B: 25-28d; C: 29-32d; D: 33-38d; E: 39-43d; F: older than 44d.
12 Bioohem. Physio!. Pflanzen, Bd. 173
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Discussion
The increase of fresh weight, dry weight and protein nitrogen takes place when celJ division is finished, and growth is the only expression of cell elongation (MANTEUFFEL et al. 1976; SMITH 1973). The observed differences to former results of our laboratory (MANTEUFFEL et at. 1976) with respect to the time course of seed development may be explained by differences of growth conditions. The incorporation of labelled leucine into the globulin fraction showed that at early stages during seed maturation the rate of vicilin synthesis is higher than the rate of legumin formation whereas at later stages the opposite is true. Quantitative determinations of the globulins of Vicia (MANTEuFFEL et at. 1976; BOULTER 1974) and Pisum (MILLERD 1975; MILLERD et at. 1975) gave similar results. The origin of the protein bodies is supposed to derive from the vacuole at least in some species, however, there is no strong evidence for this assumption (DIECKERT and DIECKERT 1976; REST and VAUGHAN 1972; SCHWARZENBACH 1971; BRIARTY et al. 1969; OPIK 1968; MIEGE and ¥ASCHERPA 1976; SlIARMA and DIECKERT 1975). The vacuole should be filled step by step by transport vesicles (UDAKA and FUKAZAWA 1977) with protein and later differentiated to aleurone g:t:ains. Until now only the beginning of this process was observed in the electron microscope and insufficient data exist for such conclusion. In Vicia (aha the formation of aleurone grains is much more complicated. In a certain stage of development a central vacuole exists, in which proteins are deposited. However, this vacuole disappears before the aleurone grains are formed by swelling of ER cisternae and evaginations of ERmembranes. This is an additional example to support the hypothesis of vacuole formation from the ER (BuvAT 1957, 1960; Poux 1962). The participation of rough ER in the synthesis of reserve proteins was shown earlier by autoradiographic investigations of tissue sections (BAILEY et al. 1970) and by serological identification of products formed in an in vitro system using isolated free and membrane-bound polysomes (MUNTZ et al. 1977). KlIoo and WOLF (197q) have demonstrated that vesicles of the ER form aleurone grains in maize. The significance of the dictyosomes on the synthesis of reserve proteins is unclear at present. In Vicia in the first stages of development of the cotyledons dictyosomes show remarkable activity until the end of the formation of aleurone grains. A direct paxticipation in the synthesis of storage proteins (e.g. uptake of the Golgi vesicles into the protein bodies) is not demonstrated in the electron microscope. Aleurone grains of Vicia contain a certain percentage of carbohydrates in form of glycoproteids (DERBYSHIRE et al. 1976). It seems possible that a part of the rese~ye proteins is stored in aleurone grains after passage the dictyosomes, where the binding of sugar to protein takes places (DIECKERT and DIECKERT 1976; UDAKA and FUKAZAWA 1977; DERBYSHIRE et at. 1976). Another possibility - the participation of the dictyosomes at the accumulation of proteins - is discussed by NEUCERE et al. (1975). However, the activity of the dictyosomes may be not correlated with the synthesesis of reserve proteins but with cell wall growth during the developmental elongation of the cotyledons (SMITlI 1973; Ro-
Protein bodies of V icia (aba seeds
179
et al. 1976). Further investigations with immunohistochemical methods are necessary to study these questions. The end of reserve protein production is connected with disappearance of the rough ER and with the formation of spherosomes being situated near the plasmalemma and surrounding amyloplasts. This phenomenon is also known from other species PhaseoZus vulgaris (OPIK 1968), Crambe abyssinica (SMITH 1974) and Bidens cernua (SIMOLA 1971). BINS ON
Acknowledgements The authors wish to thank Dr. G. SCHOLZ for valuable suggestions for the manuscript; Dr. R. MANTEUFFEL for immunoprecipitation of radioactive labeled globulins and for many helpful discussions; Mr. M. PUCHEL for the use of his radioactivity incorporating system into cotyledons; Mrs. R. HOFFMEISTER and Mrs. l. MIETHE for technical assistance.
References BAILEY, C. J., COBB., A., and BOULTER, D.: A cotyledon slice system for the electron autoradiographic study of the synthesis and intracellular transport of the seed storage protein of Vicia (aba. Planta 91),103-118 (1970). BOULTER, D.: Biosynthese, Struktur und Zusammensetzung der Samenproteine von Vicia (aba im Hinblick auf die Auslese zur Verbesserung der Proteinqualitat. Gottinger PflanzenziichterSeminar 2, 106-120 (1974). BRIARTY, L. G., COULT, D. A., and BOULTER, D.: Protein bodies of developing seeds of Vicia (aba. J. Expt. Bot. 20, 358-372 (1969). - Protein bodies of germinating seeds of Vicia (aba: changes in fine structure and biochemistry. J. Expt. Bot. 21, 513-524 (1970). BuvAT, R.: Relations entre l'ergastoplasme et l'appareil vacuolaire. C. R. Acad. Sci. (Paris) 241), 350-352 (1957). - MOUSSEAU, A.: Origine et evolution du systeme vacuolaile dans la racine de Triticum vulgare,relation avec l'ergastoplasme. C. R. Acad. Sci. (Paris) 21)1, 3051-3053 (1960). DERBYSHIRE, E., WRIGHT, D. J., and BOULTER, D.: Legumin and vicilin, storage proteins of legume seeds. Phytochemistry 11), 3-24 (1976). DIECKERT, J. W., and DIECKERT, M. C.: The chemistry and cell biology of the vacuolar proteins of seeds. J. Food Sci. 41, 475-482 (1976). ENGLEMAN, E. M.: Ontogeny of aleurone grains in cotton embryo. Amer. J. Bot. 1)3, 231-237 (1966). FLECK, A., and MUNRO, H. N.: The determination of organic nitrogen in biological materials. Clin. Chim. Acta 11, 2-12 (1965). HARRIS, N., and CHRISPEELS, M. J.: Histochemical and biochemical observations on storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans. Plant Physiol. 06, 292-299 (1975). KHOO, U., and WOLF, M. J.: Origin and development of protein granules in maize endosperm. Amer. J. Bot. 1)7, 1042-1050 (1970). MANTEUFFEL, R., MUNTZ, K., PUCHEL, M., and SCHOLZ, G.: Phase·dependen t changes of DNA, RNA an.d protein accumulation. during the ontogenesis of broad bean seeds (Vicia (aba L., var. minor). Biochem. Physiol. Pflanzen 169, 595-605 (1976). MIEGE, M.·N., and MASCHERPA, J.·M.: Isolation and analysis of protein. bodies from cotyledons of Lablab p'urpureus and Phaseolus vulgaris (Leguminoseae). Physiol. Plant. 37, 229-238 (1976). 12*
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MILLERD, A.: Biochemistry of legume seed proteins. Ann. Rev. Plant PhysioI. 26, 53-72 (1975). - SPENCER, D., DUDMAN, W. F., and STILLER, M.: Growth of immature pea cotyledons in culture. Aust. J. Plant PhysioI. 2, 51-59 (1975) ..... , . MORTON, R. K., and RAISON, J. K.: A cbmplete' intracellular unit for incorporation of amino-acid into storage protein utilizing adenosine triphosphate generated from phytate. Nature 200, 429-433 (1963). PALK, B. A., and RAISON, J. K.: Intracellular components associated with protein synthesis in developing wheat endosperm. Biochem. J. 91, 522-528 (1964). MUNTZ, K., PARTHIER, B., AURICH, 0., BASSUNER, R., MANTEUFFEL, R., PUCHEL, M., SCHMIDT, P., and SCHOLZ, G.: In vitro biosynthesis of vicilin and legumin subunits on membrane bound poly~ somes. Poster lAPP-Congress Halle 1977. NEUCERE, N. J., YATSU, L. Y., and HARRIS, J. A.: Genesis of iX-arachin synthesis in the developing peanut seed. Peanut Sci. 2, 38-41 (1975). QPIK, H.: Development of the cotyledon cell structure in ripening Phaseolus vulgaris seeds. J. Expt. Bot. 19, 64-76 (1968). ORY, R L., and HENNINGSEN, K. W.: Enzymes associated with protein bodies isolated from ungerminated barley seeds. Plant PhysioI. 44, 1488-1498 (1969). Poux, N.: Nouvelles observations sur la nature et l'origine de la membrane vacuolaire de cellules vegetales. J. de Microsc. 1, 55-66 (1962). REST, J. A., and VAUGHAN, J. G.: The development of protein and oil bodies in the seed of Sinapis alba L. Planta 105, 245-262 (1972). ROBINSON, D. G., EISINGER, W. R, and RAY, P. M.: Dynamics of the golgi system in wall matrix polysaccharide synthesis and secretion by pea cells. Ber. Dtsch. Bot. Ges. 89,147-161 (1976). SCHOLZ, G., RICHTER, J., and MANTEUFFEL, R: Studies on seed globulins from legumes. 1. Separation and purification of legumin and vicilin from Vicia (aba L. by zone precipitation. Biochem. PhysioI. Pflanzen 166, 163-172 (1974). SCHWARZENBACH, A. M.: Aleuronvakuolen und Spharosomen im Endosperm von Ricinus communis wahrend der Samenreifung und Keimung. Ber. Schweiz. Bot. Ges. 81, 70-91 (1971). SHARMA, C. B., and DIECKERT, J. W.: Isolation and partial characterization of globoids from aleurone grains of Arachis hypogaea seed. PhysioI. Plant. 33, 1-7 (1975). SMITH, D. L.: Nucleic acid, protein, and starch synthesis in developing cotyledons of Pisum arvense L. Ann. Bot. 37, 795-804 (1973). . SMITH, C. G.: The ultrastructural development of spherosomes and oil bodies in the developing embryo of Grambe abyssinica. Planta 119, 125-142 (1974). SIMOLA, L. K.: Subcellular organization of developing embryos of Bidens cernua. PhysioI. Plant. 25, 98-105 (1971). UDAKA, K., and FUKAZAWA, C.: Changes in soybean reserve protein components and protein body formation during the development. Abstract lAPP-Congress Halle (1977) 21. YATSU, L. Y.: Ultrastructure of cotyledonary tissue from Gossypium hirsutum L. seeds. J. Cell BioI. 25, 193-199 (1965).
Received March 19, 1978. Authors' address: Dr. DIETER NEUMANN, Institut fiir Biochemie der Pflanzen der AdW der DDR, DDR - 402 Halle/Saale Weinbergweg; ERNST WEBER, ZentralinstitutIiir Genetik und Kulturpflanzenforschung der AdW der DDR, DDR-4325 Gatersleben.
Formation of Protein Bodies in Ripening Seeds of Vicia faba L. D. NEUMANN and E. WEBER Institut fiir Biochemie der Pflanzen, DDR - 402 Halle (Saale) und Zentralinstitut fiir Genetik und Kulturpflanzenforschung, DDR - 4325 Gatersleben, der Akademie der Wissenschaften der DDR Key Term Index: aleurone grains, electron microscopy, development; Vicia (aba.
Summary The development of aleurone grains in ripening Vicia (aba seeds is investigated by electron microscopy and by biochemical methods. The cotyledonary cells persist in an embryonal stage during three weeks after anthesis, later they show a typical picture with stacks and strands of rough ER, stained vesicles in connection with dictyosomes and a large central vacuole. This vacuole disappears, but in the stage of reserve protein production a new vacuolar system - the aleurone grains - si formed by evagination and swelling of ER membranes. The role of the dictyosomes for the production of storage protein is discussed.
Introduction
Endosperm and cotyledons of many plant species accwnulate storage material of high uniformity in large quantities. It can be mobilized during germination and then used for the growth of the embryo in early stages of development. The origin of the storage compartment is unclear or not yet investigated in most cases. For the storage of proteins in protein bodies three compartments are discussed: plastids, vacuoles and cisternae of the endoplasmic reticulum (ER). MORTON and coworkers (1963, 1964) described protein bodies of wheat endosperm originating from plastids, since two envelope membranes could be dicriminated; however, this observation was disapproved by KHOO and WOLF (197Q). In some other species protein bodies are surrounded by only a single membrane, "very similar" to the tonoplast of Gossypium (YATSU 1965; ENGLEMAN 1966), Arachis (DIECKERT and DIECKERT 1976), Hordeum (ORY and HENNINGSEN 1969), Vitia (BRIARTY et al. 1970), Sinapis (REST and VAUGHAN 1972), Phaseolus (HARRIS and CHRISPEELS 1975). In some cases a central vacuole is filled successively with osmiophilic material identified as protein (DIECKERT and DIECKERT 1976; REST and VAUGHAN 1972). In contrast, protein bodies of other species are formed by separation from ER cisternae (KHOO and WOLF 1970). With the exception of the reserve proteins in wheat endosperm it seems obvious that protein bodies are surrounded by a single membrane only. The origin of this membrane is not clear in most cases. At present it is unknown whether the possibilities mentioned here can exist in the same tissue or in the same species.' In the present paper the development of protein bodies in Vicia faba is studied by means of electron microscopy and biochemical methods.
168
D. NEUMANN and E. WEBER
Material and Methods Plant Material Vicia (aba L., var. minor, cv. "Dornburger Ackerbohne" and cv. "Fribo" were used as materials for these experiments. During spring and summer 1976 and 1977 fully opened flowers of the 2nd, 3rd and 4th node of green house plants were labeled. After 18 days plant material was successively harvested daily until the 50th day after flowering. For analysis pods of average size containing 4 seeds were selected. Growth Oharacters
Length of seeds and cotyledons (long axis) as well as fresh and dry weights were chosen as basic morphological growth characters. Nitrogen Determinations
Whole cotyledons of early developmental stages, which were dried at 105°C, or aliquots of about 30 mg were analysed by a modified Kjeldahl method after FLECK and MUNRO (1965). 3H-Leucine Incorporation into Globulins
Freshly harvested seeds were dehulled and the testa, radicle, and plumula were dissected. Cotyledons were incubated according to PUCHEL (in preparation) for 2 h in 1 ml incubation medium of MILLERD et al. (1975) containing 50 I'ci 3H-Leu (58 Ci/mMol). The incubation flasks were shaken in the dark at 25°C and the reaction stopped by addition of ice water. Samples were stored at -20°C until used.
of Labeled Globulins Two cotyledons were homogenized with a BUhler homogenizer for 5 min with 10 ml 0.1 M phosphate buffer, pH 7.0, containing 0.5 M sodium chloride and stirred for 1 h in the same medium. Cell debris were removed by a 3000 . g centrifugation. All procedures were carried out at 4°C. The globulins legumin and viciIin were immunoprecipitated from aliquots of the extracts using an excess of monospecific antisera for legumin and vicilin, which were produced as described elsewhere (SCHOLZ et al. 1974). Extraction and Determination
Electron Microscopy
Small pieces of cotyledons were fixed with 3 % glutaraldehyde in 0.05 M cacodylate buffer pH 7.4 (2 h) and 1 % OsO, in the same buffer (1 h). For washing 0.1 mol cacodylate buffer pH 7.4 was used. Following block staining (2 % uranyl acetate in 75 % acetone, 2 h) the specimens were embedded in Araldit. Ultrathin sections cut with glass knives were stained with lead acetate and viewed in a transmission electron microscope (SEM 3/2, Werk fur Fernsehelektronik Berlin). The pronase digestions (0.01 % pronase in phosphate buffer pH 7.4, 1 h) were carried out on ultrathin sections after bleaching in 10% H2 0 2 (10 min).
Results
The increase of dry weight, fresh weight and total N of seeds and cotyledons show a typical sigmoid curve. After a lag phase of 35 days the total N content of the cotyledons increases linearly between 39 and 43 days. The rate of increase is reduced after 50 days (Fig. 1) Fresh weight and dry weights of seeds and cotyledons increase similarly (Fig. 2). The fresh weight increases mor,e in comparison with the dry weight between 28 and 32 days, but after 43 days the rate of increase of both growth parameters decreases similar to the N content. The curves seem to approach a stationary phase,
169
Protein bodies of Vicia faba seeds
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-
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Fig. 1. Total N content of the developing cotyledons of Vicia faba. Modified Kjeldahl method after FLECK and MUNRO (1965); abscissa: days after anthesis; ordinate: mg N/2 cotyledons. Fig. 2. Fresh weight and dry weight of cotyledons and seeds of Vicia faba. Absissa: days after anthesis; ordinate: mg fresh weight of seeds (-----), mg fresh weight/2 cotyledons ( - - ) , mg dry weight/2 cotyledons (-. - . -).
15 13
11
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Iii
28
iii
30 32
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34 36
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40 42
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Fig. 3. Increase in the length of developing seeds and cotyledons of Vicia faba. Abscissa: days after anthesis; ordinate: mm; seeds (---), cotyledons (- - - - -).
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D. NEUMANN and E. WEBER
which was already demonstrated by MANTEUFFEL et al. (1976) in a previous paper. The parallel course' of fT-esh weight and dry weight in later stages of development is an expression of desiccation of the ripening seeds. These changes a~e macroscopically visible in the development of the length of seeds and cotyledons (Fig. 3). They show a constant increase in length between 23 and 47 days after anthesis. After start of the desiccation of the seeds the differences become smaller, and after 48 days length of seeds and cotyledons are approximately equal. In this stage of development the N content is also approximately constant (about 12 mg N/cotyledon, that means about. 33 % ctude protein/dry weight). The seeds have reached their final length and loose their green colour. The incorporation rates of 3H-Ieucine into the globulins vicilin and legumin correspond with the linear phase of the total N-accumulation (compare Fig. 1 and Table 1). The rate of vicilin synthesis is higher than that of legumin during the first 8 days. Later the rate of legumin synthesis increases r,apidly while the rate of viciIin formation seems to decrease (Table 1). The interpretation of these differences need further experimental work, especially by immunocytochemical methods. Tabelle 1. Incorporation of 3H-leucine into storage proteins of Vicia faba during seed development. Liquid scintillation counting after immunoprecipitation with monospecific antisera of the radioactively labelled globulins vicilin and legumin. Numbers are d.p.m./aliquot days after flowering
vicilin
legumin
29 33 37 43 44 48
200 11,100 12,670 8,750 9,760 8,040
170 7,200 8,510 26,280 72,930 47,590
Electron microscopic investigations demonstrate that biochemically well characterized phases in the development of seeds are connected with typical changes in the ultrastructure of the cells. In the following the development of the cotyledons is classified in definite phases by characteristic pictures on the ultrastructural level. This classification is valid o'nly for the investigated Vicia faba val). minor, cv. "Dornburger". Other cultivars (e.g. Vicia faba var. minor, cv. "Fribo") show a similardevelopment, but a temporal shift is observed, e.g. 10 days delay for "Fribo" in comparison with "Dornburger", which is pn,obably due to different cultural conditions during spring and summer.
Phase 1 (0-24 days; Figs. 4, 5, 15A) In the first days after anthesis the metabolically active cotyledonary cells show' a dense cytoplasm and a folded plasmalemma. The cytoplasm contains small vacuoles and a large number of unstained vesicles derived from dictyosomes. The cells are connec-
Figs. 4-14. Formation of aleurone grains in ripening seeds of Vicia taba var. minor, cv. "Dornburger". Fixation: GlutaraldehydjOs04' Fig. 4. 18 days old cotyledons. Note the folded plasmalemma, dictyosomes (d), mitochondria (m), plastids (p), vacuoles (v) and endoplasmic reticulum. X 21,600. Fig. 5. 25 days old cotyledons; nucleus (n); cell wall (w); X 10,800.
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Fig.6. 29 days old cotyledons. Note the increased vacuolization and formation of rough ER. X 21,600. Fig. 7. 31 days old cotyledons. Numerous strange of rough ER, dictyosomes (d) with osmiophilic vesicles and vacuoles with dark stained material (protein-e). X 13,400.
Protein bodies of Vicia (aba seeds
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Fig. 8. 31 days old cotyledons after treatment with pronase. The light parts into the vacuole (v) are formed by enzymatic digestion of protein. X 16,000.
ted together by numerous plasmodesmata. Plastids ave very small with only some thylakoid membranes and few small starch grains.
Phase 2 (25-28 days; Figs. 6, 15B) Within few days drastic changes in the ultrastructure occur. The cells leave their embryonal stage, the number of ER membranes increases. The membranes of the endoplasmic reticulum become occupied by a large number of ribosomes. At the age of 28 days after anthesis stacks of rough ER are enriched in the cytoplasm. Products of the metabolic activity of the ER membranes cannot be localized by the electron microscope, however, this must not exclude protein synthesis, since not all proteins form an insoluble network with glutaraldehyde and can be stained by OS04·
Phase 3 (29-32 days; Figs. 7, 8, 15C) The vacuolization proceeds between 29 and 32 days, and in most of the cells only a large central vacuole is visible. Rough ER strands and stacks occupy most of the cytoplasm. Dictyosomes can be observed frequently p~oducing dark stained vesicles. On the vacuolar surface of the tonoplast and also inside the vacuoles osmiophilic ma-
Fig. 9. 35 days old cotyledons. Reserve proteins are excreted in the enlarged volume of the ER. The aleurone grains (a) may be fused together ( - - ) . X 24,000. Fig. 10-11. 35 days old cotyledons. Enlarged ER cisternae and formation of aleurone grains from the ER membranes. X 24,00; X 16,000.
Protein bodies of Vieia (aba seeds
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Fig. 12. Dictyosomes in a 36 days old cotyledon. The dictyosomes (d) forms osmiophilic vesicles. A participation of these organelles on the production of aleurone grains cannot be seen in the electron microscope. X 32,400. Fig. 13. 40 days old cotyledons. Most of the rough ER disappears and the cell is occupied by large irregular shaped aleurone grains (a). x7,200.
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Fig. 14. 44 days old cotyledons. The proportion of rough ER membranes is decreased; ER membranes divide (-) into spherosomes (s) situated around the plastids (p). X 24,000.
teJ;ial was detected. This mate:r:ial was identified as protein: Treatment of ultrathin sections with pronase removes the osmiophilic material (Fig. 8).
Phase 4 (33 -38 days; Figs. 9 -12, 15 D) Between 33 and 38 days aleurone grains are synthesized in the cotyledonary cells. This process is accompanied by dramatic changes in the ultrastructure of the cells. At first the central vacuole disappears, and within a few days aleurone grains of different size partially occupy the cytoplasmic compartment. Very often aleurone grains are surrounded by stacks of the rough ER. In some cases the formation of aleurone grains from evaginations and segregation of the ER is demonstrated (Figs. 10, 11). In the cytoplasm small vesicles without contrast or dark-stained can be observed connected to dictyosomes (Fig. 12).
Protein bodies of Vicia faba seeds
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Phase 5 (39-43 days; Figs. 13, 15 E) The formation of aleurone grains is completed in this phase. The cells contain numerous large branched aleurone grains and virtually no active dictyosomes. The proportion of rough ER membranes decreases. Phase 6 (older than 44 days; Figs. 14, 15 F) These cells show a smaller amount of rough ER in comparison with cells of the phases 3-5. It seems that smooth ER membranes divide into small vesicles and surround the plastids as spherosomes. Aleurone grains of nearly circular shape (1-2 p,m in diameter) can be observed in the cytoplasm. Fig. 15 shows schematically the formation of aleurone grains in seeds of Vicia {aha. The developing cotyledons consist of embryonic cells without specific differentiation during three weeks after anthesis (Fig. 15 A). At 24 days many stacks and strands of rough ER appear without remarkable protein accumulation at this time (Fig. 15B). Only 4 days later proteinous material is stored in the large central vacuole (Fig. 15 C). In the following time the central vacuole disappear's and a new vacuolar apparatus aleurone grains - is formed by evagination of the rough ER (Fig. 15D). Within a few days only the major quantity of the storage protein is synthesized and stored in all{lUrone grains of various size and irregular shape (Fig. 15 E). Later the rough ER disappears, spherosomes are formed from the smooth ER and the aleurone grains dissociate into small vesicles (Fig. 15F).
II
Fig. 15. Schematical hypothesis on the formation of aleurone grains in cotyledons of Vicia faba, A: 0-24d; B: 25-28d; C: 29-32d; D: 33-38d; E: 39-43d; F: older than 44d.
12 Bioohem. Physio!. Pflanzen, Bd. 173
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Discussion
The increase of fresh weight, dry weight and protein nitrogen takes place when celJ division is finished, and growth is the only expression of cell elongation (MANTEUFFEL et al. 1976; SMITH 1973). The observed differences to former results of our laboratory (MANTEUFFEL et at. 1976) with respect to the time course of seed development may be explained by differences of growth conditions. The incorporation of labelled leucine into the globulin fraction showed that at early stages during seed maturation the rate of vicilin synthesis is higher than the rate of legumin formation whereas at later stages the opposite is true. Quantitative determinations of the globulins of Vicia (MANTEuFFEL et at. 1976; BOULTER 1974) and Pisum (MILLERD 1975; MILLERD et at. 1975) gave similar results. The origin of the protein bodies is supposed to derive from the vacuole at least in some species, however, there is no strong evidence for this assumption (DIECKERT and DIECKERT 1976; REST and VAUGHAN 1972; SCHWARZENBACH 1971; BRIARTY et al. 1969; OPIK 1968; MIEGE and ¥ASCHERPA 1976; SlIARMA and DIECKERT 1975). The vacuole should be filled step by step by transport vesicles (UDAKA and FUKAZAWA 1977) with protein and later differentiated to aleurone g:t:ains. Until now only the beginning of this process was observed in the electron microscope and insufficient data exist for such conclusion. In Vicia (aha the formation of aleurone grains is much more complicated. In a certain stage of development a central vacuole exists, in which proteins are deposited. However, this vacuole disappears before the aleurone grains are formed by swelling of ER cisternae and evaginations of ERmembranes. This is an additional example to support the hypothesis of vacuole formation from the ER (BuvAT 1957, 1960; Poux 1962). The participation of rough ER in the synthesis of reserve proteins was shown earlier by autoradiographic investigations of tissue sections (BAILEY et al. 1970) and by serological identification of products formed in an in vitro system using isolated free and membrane-bound polysomes (MUNTZ et al. 1977). KlIoo and WOLF (197q) have demonstrated that vesicles of the ER form aleurone grains in maize. The significance of the dictyosomes on the synthesis of reserve proteins is unclear at present. In Vicia in the first stages of development of the cotyledons dictyosomes show remarkable activity until the end of the formation of aleurone grains. A direct paxticipation in the synthesis of storage proteins (e.g. uptake of the Golgi vesicles into the protein bodies) is not demonstrated in the electron microscope. Aleurone grains of Vicia contain a certain percentage of carbohydrates in form of glycoproteids (DERBYSHIRE et al. 1976). It seems possible that a part of the rese~ye proteins is stored in aleurone grains after passage the dictyosomes, where the binding of sugar to protein takes places (DIECKERT and DIECKERT 1976; UDAKA and FUKAZAWA 1977; DERBYSHIRE et at. 1976). Another possibility - the participation of the dictyosomes at the accumulation of proteins - is discussed by NEUCERE et al. (1975). However, the activity of the dictyosomes may be not correlated with the synthesesis of reserve proteins but with cell wall growth during the developmental elongation of the cotyledons (SMITlI 1973; Ro-
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et al. 1976). Further investigations with immunohistochemical methods are necessary to study these questions. The end of reserve protein production is connected with disappearance of the rough ER and with the formation of spherosomes being situated near the plasmalemma and surrounding amyloplasts. This phenomenon is also known from other species PhaseoZus vulgaris (OPIK 1968), Crambe abyssinica (SMITH 1974) and Bidens cernua (SIMOLA 1971). BINS ON
Acknowledgements The authors wish to thank Dr. G. SCHOLZ for valuable suggestions for the manuscript; Dr. R. MANTEUFFEL for immunoprecipitation of radioactive labeled globulins and for many helpful discussions; Mr. M. PUCHEL for the use of his radioactivity incorporating system into cotyledons; Mrs. R. HOFFMEISTER and Mrs. l. MIETHE for technical assistance.
References BAILEY, C. J., COBB., A., and BOULTER, D.: A cotyledon slice system for the electron autoradiographic study of the synthesis and intracellular transport of the seed storage protein of Vicia (aba. Planta 91),103-118 (1970). BOULTER, D.: Biosynthese, Struktur und Zusammensetzung der Samenproteine von Vicia (aba im Hinblick auf die Auslese zur Verbesserung der Proteinqualitat. Gottinger PflanzenziichterSeminar 2, 106-120 (1974). BRIARTY, L. G., COULT, D. A., and BOULTER, D.: Protein bodies of developing seeds of Vicia (aba. J. Expt. Bot. 20, 358-372 (1969). - Protein bodies of germinating seeds of Vicia (aba: changes in fine structure and biochemistry. J. Expt. Bot. 21, 513-524 (1970). BuvAT, R.: Relations entre l'ergastoplasme et l'appareil vacuolaire. C. R. Acad. Sci. (Paris) 241), 350-352 (1957). - MOUSSEAU, A.: Origine et evolution du systeme vacuolaile dans la racine de Triticum vulgare,relation avec l'ergastoplasme. C. R. Acad. Sci. (Paris) 21)1, 3051-3053 (1960). DERBYSHIRE, E., WRIGHT, D. J., and BOULTER, D.: Legumin and vicilin, storage proteins of legume seeds. Phytochemistry 11), 3-24 (1976). DIECKERT, J. W., and DIECKERT, M. C.: The chemistry and cell biology of the vacuolar proteins of seeds. J. Food Sci. 41, 475-482 (1976). ENGLEMAN, E. M.: Ontogeny of aleurone grains in cotton embryo. Amer. J. Bot. 1)3, 231-237 (1966). FLECK, A., and MUNRO, H. N.: The determination of organic nitrogen in biological materials. Clin. Chim. Acta 11, 2-12 (1965). HARRIS, N., and CHRISPEELS, M. J.: Histochemical and biochemical observations on storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans. Plant Physiol. 06, 292-299 (1975). KHOO, U., and WOLF, M. J.: Origin and development of protein granules in maize endosperm. Amer. J. Bot. 1)7, 1042-1050 (1970). MANTEUFFEL, R., MUNTZ, K., PUCHEL, M., and SCHOLZ, G.: Phase·dependen t changes of DNA, RNA an.d protein accumulation. during the ontogenesis of broad bean seeds (Vicia (aba L., var. minor). Biochem. Physiol. Pflanzen 169, 595-605 (1976). MIEGE, M.·N., and MASCHERPA, J.·M.: Isolation and analysis of protein. bodies from cotyledons of Lablab p'urpureus and Phaseolus vulgaris (Leguminoseae). Physiol. Plant. 37, 229-238 (1976). 12*
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MILLERD, A.: Biochemistry of legume seed proteins. Ann. Rev. Plant PhysioI. 26, 53-72 (1975). - SPENCER, D., DUDMAN, W. F., and STILLER, M.: Growth of immature pea cotyledons in culture. Aust. J. Plant PhysioI. 2, 51-59 (1975) ..... , . MORTON, R. K., and RAISON, J. K.: A cbmplete' intracellular unit for incorporation of amino-acid into storage protein utilizing adenosine triphosphate generated from phytate. Nature 200, 429-433 (1963). PALK, B. A., and RAISON, J. K.: Intracellular components associated with protein synthesis in developing wheat endosperm. Biochem. J. 91, 522-528 (1964). MUNTZ, K., PARTHIER, B., AURICH, 0., BASSUNER, R., MANTEUFFEL, R., PUCHEL, M., SCHMIDT, P., and SCHOLZ, G.: In vitro biosynthesis of vicilin and legumin subunits on membrane bound poly~ somes. Poster lAPP-Congress Halle 1977. NEUCERE, N. J., YATSU, L. Y., and HARRIS, J. A.: Genesis of iX-arachin synthesis in the developing peanut seed. Peanut Sci. 2, 38-41 (1975). QPIK, H.: Development of the cotyledon cell structure in ripening Phaseolus vulgaris seeds. J. Expt. Bot. 19, 64-76 (1968). ORY, R L., and HENNINGSEN, K. W.: Enzymes associated with protein bodies isolated from ungerminated barley seeds. Plant PhysioI. 44, 1488-1498 (1969). Poux, N.: Nouvelles observations sur la nature et l'origine de la membrane vacuolaire de cellules vegetales. J. de Microsc. 1, 55-66 (1962). REST, J. A., and VAUGHAN, J. G.: The development of protein and oil bodies in the seed of Sinapis alba L. Planta 105, 245-262 (1972). ROBINSON, D. G., EISINGER, W. R, and RAY, P. M.: Dynamics of the golgi system in wall matrix polysaccharide synthesis and secretion by pea cells. Ber. Dtsch. Bot. Ges. 89,147-161 (1976). SCHOLZ, G., RICHTER, J., and MANTEUFFEL, R: Studies on seed globulins from legumes. 1. Separation and purification of legumin and vicilin from Vicia (aba L. by zone precipitation. Biochem. PhysioI. Pflanzen 166, 163-172 (1974). SCHWARZENBACH, A. M.: Aleuronvakuolen und Spharosomen im Endosperm von Ricinus communis wahrend der Samenreifung und Keimung. Ber. Schweiz. Bot. Ges. 81, 70-91 (1971). SHARMA, C. B., and DIECKERT, J. W.: Isolation and partial characterization of globoids from aleurone grains of Arachis hypogaea seed. PhysioI. Plant. 33, 1-7 (1975). SMITH, D. L.: Nucleic acid, protein, and starch synthesis in developing cotyledons of Pisum arvense L. Ann. Bot. 37, 795-804 (1973). . SMITH, C. G.: The ultrastructural development of spherosomes and oil bodies in the developing embryo of Grambe abyssinica. Planta 119, 125-142 (1974). SIMOLA, L. K.: Subcellular organization of developing embryos of Bidens cernua. PhysioI. Plant. 25, 98-105 (1971). UDAKA, K., and FUKAZAWA, C.: Changes in soybean reserve protein components and protein body formation during the development. Abstract lAPP-Congress Halle (1977) 21. YATSU, L. Y.: Ultrastructure of cotyledonary tissue from Gossypium hirsutum L. seeds. J. Cell BioI. 25, 193-199 (1965).
Received March 19, 1978. Authors' address: Dr. DIETER NEUMANN, Institut fiir Biochemie der Pflanzen der AdW der DDR, DDR - 402 Halle/Saale Weinbergweg; ERNST WEBER, ZentralinstitutIiir Genetik und Kulturpflanzenforschung der AdW der DDR, DDR-4325 Gatersleben.