Planta
Planta 150, 419-425 (1980)
9 by Springer-Verlag 1980
Immunocytochemical Localization of Reserve Protein in the Endoplasmic Reticulum of Developing Bean (Phaseolus vulgaris) Cotyledons Bruno Baumgartner*, K.T. Tokuyasu, and Maarten J. Chrispeels** Department of Biology~ C-016, University of California/San Diego, La Jolla, CA 92093, USA
Abstract. The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at - 8 0 ~ C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 gin. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobulin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G. Key words: Cotyledons Endoplasmic reticulum Ferritin labeling - Immunocytochemistry - Phaseolus - Protein (reserve) Reserve protein.
* Present address." Oberer Stiinziweg 10, CH-8942 Oberriedem Switzerland ** To whom requests for reprints should be addressed Abbreviations. ER=endoplasmic reticulum; IgG=immunoglobu-
lin G
Introduction In the common bean (Phaseolus vulgaris L.) some 50% of the total protein present in the mature seed is the 7S storage protein phaseolin 1. Phaseolin is synthesised during seed maturation and accumulates in the storage parenchyma cells of the cotyledons. Within these cells it is contained in protein bodies, large spherical organelles consisting of an amorphous protein matrix surrounded by a limiting membrane. The storage parenchyma cells of the cotyledons have a very extensive rough endoplasmic reticulum (ER) (Opik 1968; Bain and Mercer 1966) and cotyledon development is accompanied by a dramatic increase in the cytoplasmic volume occupied by the rough ER (Briarty 1973). Autoradiography of Vicafaba cotyledons showed the rough ER to be a major site of amino-acid incorporation and protein synthesis, and it was suggested by Bailey et al. (1970) that the rough ER is the site of storage-protein biosynthesis. This suggestion was confirmed by the finding that the polypeptides of the reserve proteins are synthesised in vitro by polysomes derived from the rough ER, but not by free polysomes (Bollini and Chrispeels 1979). The reserve proteins of legumes are glycoproteins containing mannose and N-acetylglucosamine, and in Pisum sativum cotyledons the glycosyltransferases involved in the transfer of glycosylresidues to polypeptide chains have been shown to be associated with the ER (Nagahashi and Beevers 1978). Together, these observations indicate that the reserve proteins may be sequestered in the ER after they have been synthesised, and that the ER may function in their transport to the protein bodies. We have used the 1 In agreement with many other workers the term phaseolin is used to describe the most abundant reserve protein in the cotyledons of Phaseolus vulgaris. We have used the term vicilin in earlier publications. The same protein is also known as globulin G1 and glycoprotein II
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B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
Figs. 1 and 2. Ultrathin frozen sections of cotyledon of P. vulgar& showing ultrastructural preservation after fixation with glutaraldehyde, paraformaldehyde and acrolein. The sections were stained with uranyl acetate (neutral and acidic). Nu, nucleus; PB, protein body; Go, Golgi apparatus; ER, endoplasmic reticulum; V, vesicles. Fig. 1 : • 13,500, b a r = 1 ~tm; Fig. 2: • 48,600, b a r = 1 gm
B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
421
affinity chromatography on rabbit IgG coupled to Ultrogel AcA 22 (LKB, Stockholm, Sweden) activated with glutaraldehyde (Ternyck and Avrameas 1972). The activated gel was incubated with the anti-rabbit IgG at room temperature for 1 h. After intensive washing with phosphate-buffered saline (BBS), the active antibodies were eluted with 0.2 M HC1 adjusted to pH 2.2 with 2 M glycine. The eluate was immediately neutralized with 1 M Na2HPO4. The active IgG fraction was conjugated with ferritin (EM-grade; Polysciences, Warrington, Pa., USA) after glutaraldehyde activation according to the method described by Kishida et al. (1975). Antibody concentrations for the immunostaining of ultrathin frozen sections were 0.02-0.2 mg/ml.
Fig. 3. Crossed immunoelectrophoresis of phaseolin and anti-phaseolin IgG. Phaseolin was placed in the circular well and electrophoresis was carried out at 10 V/cm for 60 min. Electrophoresis in the second dimension was at 2 V/cm for 65 h into an agarose gel containing anti-vicilin IgG
technique of indirect immunolabelling of ultrathin f r o z e n s e c t i o n s to l o c a l i z e p h a s e o l i n at t h e u l t r a s t r u c t u r a l level a n d o b t a i n e d d a t a i n d i c a t i n g t h a t p h a s e o l i n is i n d e e d s e q u e s t e r e d in t h e c i s t e r n a e o f t h e E R as well as in E R vesicles.
Material and Methods Plants. Seeds of Phaseolus vulgaris L., cv. Greensleeves, were purchased from W. Atlee Burpee Co., Riverside, Cal., USA, and the plants were grown as described in Bollini and Chrispeels (1979), in a greenhouse. Purification of Phaseolin, Preparation of the Antibodies, and Crossed Immunoelectrophoresis. Phaseolin was purified from dry mature beans as described previously, in Bollini and Chrispeels (1978). Antibodies were produced and the immunoglobulin G (IgG) fraction purified as also described in Bollini and Chrispeels (1979). Crossed immunoelectrophoresis was carried out by a modification of the procedure of Laurell (1965). The electrophoresis buffer was barbital-glycine-tris(hydroxymethyl)-aminomethane (Tris), pH 8.6, ionic strength 0.04. Glass plates measured 85 x 95 mm 2. Gels consisted of 1% agarose (agarose L; LKB, Bromma, Sweden) in electrophoresis buffer. A 10-ml gel was cast on the plate and 10 gl of phaseolin (5 mg/ml) placed in a circular well. Phaseolin was solubilized in 0.1 M Tris-glycine, pH 8.6, containing 4% Triton X-100 (Sigma Chemical Co., St. Louis, Mo., USA). Electrophoresis in the first dimension was at 10 V/cm for 60 rain in a water-cooled multiphor electrophoresis cell (LKB). An agarose strip (20 mm x 85 mm) containing the electrophoresed proteins was cut from the first gel and placed on a new glass plate next to a second gel containing 100 gl of the anti-phaseolin IgG in 8 ml of 1% agarose in electrophoresis buffer. Electrophoresis in the second dimension was carried out for 48-72 h at 2 V/cm The gels were washed for 3 h in 0.3 M NaC1, then washed for 3 h in H20, dried, and stained with Coomassie brilliant blue R-250 (ICI United States. Wilmington, Del., USA) (Weeke 1973).
Preparation of Ferritin-Conjugated Antibodies. Goat antibodies against rabbit IgG (Nordic, Antwerp, Belgium) were purified by
Tissue Processing. Tissue was obtained form seeds weighing 250300 mg. At this stage (20-25 d after pollination) phaseolin synthesis and accumulation is most rapid. Tissue blocks of approx. 0.2 mm 3 were cut directly in PBS containing 0.1 M sucrose, 4% paraformaldehyde, 0.75% glutaraldehyde and 0.75% acrolein. The tissue was kept in this solution at 3~ for 12 h. The fixed blocks were then transferred to PBS containing 1.0 M sucrose, 4% paraformaldehyde, 0.75% glutaraldehyde and 0.75% acrolein, and infused with this solution for 20 h at 3~ C. The tissue blocks were mounted on copper rods, frozen in liquid nitrogen, and immediately transferred into the precooled cryokit bowl of a Sorvall Porter-Blum MT-2B ultramicrotome (Du Pont Instruments-Sorvall, Newton, Conn., USA). Ultrathin sections were cut from the frozen tissue blocks at - 8 0 ~ C. The sections were picked up with a small wire loop containing a semi-frozen drop of a mixture of 2.0 M sucrose and 0.75% gelatin in 0.0l M phosphate buffer, pH 7.4, and transferred to copper grids covered with ionized formvar-carbon films. The grids were placed on 1% gelatin, 0.3% agarose in 0.01 M phosphate buffer, pH 7.4, with the absorbed sections towards the solution. The indirect immunolabelling with antibodies against phaseolin followed by ferritin-conjugated goat anti-rabbit IgG was performed as described by Tokuyasu and Singer (1976). The sections were positively stained for I0 min with 2% uranyl acetate brought to pH 7.1 (Tokuyasu 1978) and then for 10 rain in 0.005% uranyl acetate, pH 4.0, in 0.2% methylcellulose (Fisher Scientific, Fairlawn, N.J., USA) and 0.8% Carbowax (polyethyleneglycol, waxy; Polysciences). The sections were then embedded in a thin film of methylcellulose and Carbowax (Tokuyasu 1980), and examined in a Philips (Eindhoven, The Netherlands) EM-300 electron microscope at 60 kV.
Results and Discussion Preservation o f Ultrastructure in Frozen Sections. T h e u l t r a s t r u c t u r e o f t h e s t o r a g e - p a r e n c h y m a cells o f dev e l o p i n g l e g u m e c o t y l e d o n s has b e e n d e s c r i b e d in several p a p e r s (e.g. B a i n a n d M e r c e r 1966; {)pik 1968; B r i a r t y et al. 1969; H a r r i s a n d B o u l t e r 1976; H a r r i s 1979) a n d will n o t b e d o c u m e n t e d in d e t a i l here. T h e cells o f P. vulgaris c o t y l e d o n s h a v e l a r g e s t a r c h g r a i n s , n u m e r o u s p r o t e i n b o d i e s m e a s u r i n g 1-10 g m in d i a m eter, a v e r y e x t e n s i v e n e t w o r k o f r o u g h E R c i s t e r n a e , a n d all o t h e r o r g a n e l l e s n o r m a l l y p r e s e n t in p h y s i o l o g i c a l l y a c t i v e cells. A n e x a m i n a t i o n o f t h i n s e c t i o n s of frozen cotyledons showed that ultracryotomy could be r e a d i l y a p p l i e d to t h e s e p l a n t o r g a n s , a n d t h a t t h e r e was e x c e l l e n t p r e s e r v a t i o n o f u l t r a s t r u c t u r e if t h e s p e c i m e n s w e r e f i x e d in a m i x t u r e o f f o r m a l d e h y d e ( 4 % , w/v), g l u t a r a l d e h y d e ( 0 . 7 5 % , v/v) a n d a c r o l e i n
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B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
Figs. 4 and 5. Ultrathin frozen sections of cotyledon of P. vulgaris labeled with rabbit anti-phaseolin IgG (0.2 mg/ml) and then with goat-anti-rabbit IgG conjugated to ferritin. Note the very dense distribution of ferritin particles over the protein bodies (PB), vesicles (V) and ER. Fig. 4: x 66,000, bar=0.5 p,m; Fig. 5: x 54,000, bar=0.5 gm
B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
423
Figs. 6 and 7. Ultrathin frozen sections of cotyledon of P. vulgaris labeled as in Figs. 4 and 5, except that the concentration of rabbit anti-phaseolin IgG was only 0.02 mg/ml. A small n u m b e r of ferritin particles are now bound, but specificity is greater here than in Fig. 4. ER cisternae are labeled, as are vesicles closely associated with Golgi apparatus (Go); mitochondria (m) are not labeled. x 100,000, b a r = 0 . 5 p,m
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B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
(0.75%, v/v). Fixation in formaldehyde alone or in formaldehyde with acrolein resulted in poor preservation of ultrastructure and/or poor sectioning of the frozen specimens. Ultrastructural preservation of the cytoplasmic organelles in storage parenchyma cells is shown in Fig. 1. The cell contains protein bodies and a very extensive ER, as well as numerous dense vesicles. The ER cisternae are known to be covered with ribosomes (Opik 1968; Briarty 1973) and polysomes obtained from isolated ER are engaged in the biosynthesis of phaseolin (Bollini and Chrispeels 1979). Dictyosomes, i.e., Golgi apparatuses, are also clearly visible in the frozen sections (Fig. 2) although we did not observe many clearly recognizable dictyosomes consisting of a stack of cisternae with associated secretory vesicles. Mitochondria were more difficult to identify, especially at the lower magnification shown in Fig. 1, and were easily confused with small protein bodies. At higher magnification the non-uniformity in the internal structure based on the presence of cristae, made it possible to identify mitochondria (see Figs. 6, 7). Characterization of the Antibodies. Phaseolin was purified in its 18S form on sucrose gradients at pH 4.5. When subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis this pure phaseolin contained four polypeptides in the 50,000 molecularweight range (Bollini and Chrispeels 1979). No contaminants were visible on overloaded gels. The purified phasolin was used as antigen and the resulting rabbit antiserum fractionated to yield an IgG fraction. The purity of the antigen was also checked by crossed immunoelectrophoresis. A single precipitation arc was formed (Fig. 3) indicating that the antigen (phaseolin) was indeed a pure protein. The IgG fraction was used for the indirect immunolabelling experiments described below. Localization of Phaseolin. Using a high concentration of IgG (0.2 mg/ml) we observed very heavy labeling with ferritin particles over the protein bodies, the cisternae of the ER and associated ER vesicles (Figs. 4, 5); some ferritin particles could also be seen over the electron-translucent areas of cytoplasm apparently devoid of ER or proteins bodies. This low degree of specificity was considered to be a consequence of the high concentration of IgG. Using a low concentration of anti-phaseolin IgG (0.02 mg/ml) we observed lighter labeling of the same structures (protein bodies, ER cisternae and vesicles). The low concentration, on the other hand, resulted in greater specificity of the antibody-antigen reaction; the number of ferritin particles bound to the tissue section was much smaller, but they were exclusively found to be associated with protein bodies, ER cister-
nae and ER vesicles (Figs. 6, 7). In the top-right-hand corner of Fig. 7 ferritin particles are present over vesicles found in close proximity to the dictyosome (arrows) but not over other areas of the organelle. Whether this represents labeling of the dictyosome or labeling of a nearby ER cisterna is not clear. The mitochondria and the cytosol are not labeled with ferritin particles in Figs. 6 and 7, indicating the high specificity of labeling at the lowIgG concentration. Control experiments were carried out by treating section with pre-immune rabbit IgG followed by ferritinconjugated goat anti-rabbit IgG. Very few ferritin particles were visible on sections treated in this manner (data not shown), indicating that the pre-immune rabbit IgG did not bind non-specifically to the sections. The subcellular localization of phaseolin was also examined in the cotyledons of 2-d-old seedlings. At that time in cotyledon development, ferritin particles were found only over the protein bodies but not over the ER (data not shown). Such an observation is consistent with the finding that phaseolin synthesis stops towards the end of seed maturation (Sun et al. 1978; Bollini and Chrispeels 1979). Conclusion. The results presented here show that the technique of cutting ultrathin sections from frozen specimens and labeling these sections with ferritinconjugated antibodies is applicable to plant cells; ultrastructural preservation was found to be excellent and the various organelles clearly recognizable after staining. The storage protein phaseolin is localized not only in the protein bodies, but also in the cisternae and vesicles of the ER. We postulate that phaseolin is sequestered in the ER after it is synthesized by ER-bound polysomes (Bollini and Chrispeels 1979) and that the ER functions in the transport of phaseolin to the protein bodies. There is at present insufficient evidence to decide whether or not the Golgi apparatus also plays a role in this transport. Supported by grants from the National ScienceFoundation (Metabolic Biology)and North-Atlantic Treaty Organization to M.J.C., and by a grant from the U.S. Public Health Service to S.J. Singer and K.T.T.
References Bailey, C.J., Cobb, A., Boulter, D. (1970)A cotyledon slice system for the electron autoradiographic study of the synthesis and intracellular transport of seed storage protein of Vicia faba. Planta 95, 103-118 Bain, J.M., Mercer, F.V. (1966) Subcellular organisation of the developing cotyledons of Pisum sativum. Aust. J. Biol. Sci. 19, 49-67 Bollini, R., Chrispeels, M.J. (1978) Characterization and subcellu-
B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons lar localization of vicilin and phytohemagglutinin, the two major reserve proteins of Phaseolus vulgaris L. Planta 142, 291-298 Bollini, R., Chrispeels, M.J. (1979) The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons. Planta 146, 487-50i Briarty, L.G. (1973) Stereology in seed development studies: Some preliminary work. Caryologia 25, Suppl. 289-301 Briarty, L.G., Coult, D.A., Boulter, D. (1969) Protein bodies of developing seeds of Viciafaba. J. Exp. Bot. 20, 358 372 Harris, N. (1979) Endoplasmic reticulum in developing seeds of Vicia faba. A high voltage electron microscopic study. Planta 146, 63-69 Harris, N., Boulter D. (1976) Protein body formation in cotyledons of developing cowpea (Vigna unguiculata) seeds. Ann. Bot. 40, 739 744 Kishida, Y., Olsen, B.R., Berg, R.A., Prockop, D.J. (1975) Two improved methods for preparing ferritin-protein conjugates for electron microscopy. J. Cell Biol. 64, 331 339 Laurell, C.-B. (1965) Antigen antibody crossed electrophoresis. Anal. Biochem. 10, 358 361 Nagahashi, J., Beevers, L. (t978) Subcellular localization of glycosyltransferases involved in glycoprotein biosynthesis in the cotyledons of Pisurn sativurn L. Plant Physiol. 61, 451-459
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Opik, H. (1968) Development of cotyledon cell structure in ripening Phaseolus vulgaris seeds. J. Exp. Bot. 19, 64-76 Sun, S.M., Mutschler, M.A., Bliss, F.A., Hall, T.C. (1978) Protein synthesis and accumulation in bean cotyledons during growth. Plant Physiol 61, 918-923 Ternyck, T., Avrameas, S. (1972) Polyacrylamide-protein immunoadsorbents prepared with glutaraldehyde. FEBS Lett. 23, 24-28 Tokuyasu, K.T. (1978) A study of positive staining of ultrathin frozen sections. J. Ultrastruct. Res. 63, 287-307 Tokuyasu, K.T. (i980) Adsorption staining method for ultrathin frozen sections. Proc. 38th Ann. Meet. Electr. Micr. Soc. America, pp. 760 763 Tokuyasu, K.T., Singer, S.J. (1976) Improved procedures for immunoferritin labelling of ultrathin frozen sections. J. Cell Biol. 71, 894-906 Weeke, B. (1973) Rocket immunoelectrophoresis. Crossed immunoelectrophoresis. Scand. J. Immunol. 2, Suppl. 1, 37-56
Received 23 July; accepted 22 September 1980
Planta 150, 419-425 (1980)
9 by Springer-Verlag 1980
Immunocytochemical Localization of Reserve Protein in the Endoplasmic Reticulum of Developing Bean (Phaseolus vulgaris) Cotyledons Bruno Baumgartner*, K.T. Tokuyasu, and Maarten J. Chrispeels** Department of Biology~ C-016, University of California/San Diego, La Jolla, CA 92093, USA
Abstract. The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at - 8 0 ~ C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 gin. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobulin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G. Key words: Cotyledons Endoplasmic reticulum Ferritin labeling - Immunocytochemistry - Phaseolus - Protein (reserve) Reserve protein.
* Present address." Oberer Stiinziweg 10, CH-8942 Oberriedem Switzerland ** To whom requests for reprints should be addressed Abbreviations. ER=endoplasmic reticulum; IgG=immunoglobu-
lin G
Introduction In the common bean (Phaseolus vulgaris L.) some 50% of the total protein present in the mature seed is the 7S storage protein phaseolin 1. Phaseolin is synthesised during seed maturation and accumulates in the storage parenchyma cells of the cotyledons. Within these cells it is contained in protein bodies, large spherical organelles consisting of an amorphous protein matrix surrounded by a limiting membrane. The storage parenchyma cells of the cotyledons have a very extensive rough endoplasmic reticulum (ER) (Opik 1968; Bain and Mercer 1966) and cotyledon development is accompanied by a dramatic increase in the cytoplasmic volume occupied by the rough ER (Briarty 1973). Autoradiography of Vicafaba cotyledons showed the rough ER to be a major site of amino-acid incorporation and protein synthesis, and it was suggested by Bailey et al. (1970) that the rough ER is the site of storage-protein biosynthesis. This suggestion was confirmed by the finding that the polypeptides of the reserve proteins are synthesised in vitro by polysomes derived from the rough ER, but not by free polysomes (Bollini and Chrispeels 1979). The reserve proteins of legumes are glycoproteins containing mannose and N-acetylglucosamine, and in Pisum sativum cotyledons the glycosyltransferases involved in the transfer of glycosylresidues to polypeptide chains have been shown to be associated with the ER (Nagahashi and Beevers 1978). Together, these observations indicate that the reserve proteins may be sequestered in the ER after they have been synthesised, and that the ER may function in their transport to the protein bodies. We have used the 1 In agreement with many other workers the term phaseolin is used to describe the most abundant reserve protein in the cotyledons of Phaseolus vulgaris. We have used the term vicilin in earlier publications. The same protein is also known as globulin G1 and glycoprotein II
0032-0935/80/0150/0419/$01.40
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B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
Figs. 1 and 2. Ultrathin frozen sections of cotyledon of P. vulgar& showing ultrastructural preservation after fixation with glutaraldehyde, paraformaldehyde and acrolein. The sections were stained with uranyl acetate (neutral and acidic). Nu, nucleus; PB, protein body; Go, Golgi apparatus; ER, endoplasmic reticulum; V, vesicles. Fig. 1 : • 13,500, b a r = 1 ~tm; Fig. 2: • 48,600, b a r = 1 gm
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affinity chromatography on rabbit IgG coupled to Ultrogel AcA 22 (LKB, Stockholm, Sweden) activated with glutaraldehyde (Ternyck and Avrameas 1972). The activated gel was incubated with the anti-rabbit IgG at room temperature for 1 h. After intensive washing with phosphate-buffered saline (BBS), the active antibodies were eluted with 0.2 M HC1 adjusted to pH 2.2 with 2 M glycine. The eluate was immediately neutralized with 1 M Na2HPO4. The active IgG fraction was conjugated with ferritin (EM-grade; Polysciences, Warrington, Pa., USA) after glutaraldehyde activation according to the method described by Kishida et al. (1975). Antibody concentrations for the immunostaining of ultrathin frozen sections were 0.02-0.2 mg/ml.
Fig. 3. Crossed immunoelectrophoresis of phaseolin and anti-phaseolin IgG. Phaseolin was placed in the circular well and electrophoresis was carried out at 10 V/cm for 60 min. Electrophoresis in the second dimension was at 2 V/cm for 65 h into an agarose gel containing anti-vicilin IgG
technique of indirect immunolabelling of ultrathin f r o z e n s e c t i o n s to l o c a l i z e p h a s e o l i n at t h e u l t r a s t r u c t u r a l level a n d o b t a i n e d d a t a i n d i c a t i n g t h a t p h a s e o l i n is i n d e e d s e q u e s t e r e d in t h e c i s t e r n a e o f t h e E R as well as in E R vesicles.
Material and Methods Plants. Seeds of Phaseolus vulgaris L., cv. Greensleeves, were purchased from W. Atlee Burpee Co., Riverside, Cal., USA, and the plants were grown as described in Bollini and Chrispeels (1979), in a greenhouse. Purification of Phaseolin, Preparation of the Antibodies, and Crossed Immunoelectrophoresis. Phaseolin was purified from dry mature beans as described previously, in Bollini and Chrispeels (1978). Antibodies were produced and the immunoglobulin G (IgG) fraction purified as also described in Bollini and Chrispeels (1979). Crossed immunoelectrophoresis was carried out by a modification of the procedure of Laurell (1965). The electrophoresis buffer was barbital-glycine-tris(hydroxymethyl)-aminomethane (Tris), pH 8.6, ionic strength 0.04. Glass plates measured 85 x 95 mm 2. Gels consisted of 1% agarose (agarose L; LKB, Bromma, Sweden) in electrophoresis buffer. A 10-ml gel was cast on the plate and 10 gl of phaseolin (5 mg/ml) placed in a circular well. Phaseolin was solubilized in 0.1 M Tris-glycine, pH 8.6, containing 4% Triton X-100 (Sigma Chemical Co., St. Louis, Mo., USA). Electrophoresis in the first dimension was at 10 V/cm for 60 rain in a water-cooled multiphor electrophoresis cell (LKB). An agarose strip (20 mm x 85 mm) containing the electrophoresed proteins was cut from the first gel and placed on a new glass plate next to a second gel containing 100 gl of the anti-phaseolin IgG in 8 ml of 1% agarose in electrophoresis buffer. Electrophoresis in the second dimension was carried out for 48-72 h at 2 V/cm The gels were washed for 3 h in 0.3 M NaC1, then washed for 3 h in H20, dried, and stained with Coomassie brilliant blue R-250 (ICI United States. Wilmington, Del., USA) (Weeke 1973).
Preparation of Ferritin-Conjugated Antibodies. Goat antibodies against rabbit IgG (Nordic, Antwerp, Belgium) were purified by
Tissue Processing. Tissue was obtained form seeds weighing 250300 mg. At this stage (20-25 d after pollination) phaseolin synthesis and accumulation is most rapid. Tissue blocks of approx. 0.2 mm 3 were cut directly in PBS containing 0.1 M sucrose, 4% paraformaldehyde, 0.75% glutaraldehyde and 0.75% acrolein. The tissue was kept in this solution at 3~ for 12 h. The fixed blocks were then transferred to PBS containing 1.0 M sucrose, 4% paraformaldehyde, 0.75% glutaraldehyde and 0.75% acrolein, and infused with this solution for 20 h at 3~ C. The tissue blocks were mounted on copper rods, frozen in liquid nitrogen, and immediately transferred into the precooled cryokit bowl of a Sorvall Porter-Blum MT-2B ultramicrotome (Du Pont Instruments-Sorvall, Newton, Conn., USA). Ultrathin sections were cut from the frozen tissue blocks at - 8 0 ~ C. The sections were picked up with a small wire loop containing a semi-frozen drop of a mixture of 2.0 M sucrose and 0.75% gelatin in 0.0l M phosphate buffer, pH 7.4, and transferred to copper grids covered with ionized formvar-carbon films. The grids were placed on 1% gelatin, 0.3% agarose in 0.01 M phosphate buffer, pH 7.4, with the absorbed sections towards the solution. The indirect immunolabelling with antibodies against phaseolin followed by ferritin-conjugated goat anti-rabbit IgG was performed as described by Tokuyasu and Singer (1976). The sections were positively stained for I0 min with 2% uranyl acetate brought to pH 7.1 (Tokuyasu 1978) and then for 10 rain in 0.005% uranyl acetate, pH 4.0, in 0.2% methylcellulose (Fisher Scientific, Fairlawn, N.J., USA) and 0.8% Carbowax (polyethyleneglycol, waxy; Polysciences). The sections were then embedded in a thin film of methylcellulose and Carbowax (Tokuyasu 1980), and examined in a Philips (Eindhoven, The Netherlands) EM-300 electron microscope at 60 kV.
Results and Discussion Preservation o f Ultrastructure in Frozen Sections. T h e u l t r a s t r u c t u r e o f t h e s t o r a g e - p a r e n c h y m a cells o f dev e l o p i n g l e g u m e c o t y l e d o n s has b e e n d e s c r i b e d in several p a p e r s (e.g. B a i n a n d M e r c e r 1966; {)pik 1968; B r i a r t y et al. 1969; H a r r i s a n d B o u l t e r 1976; H a r r i s 1979) a n d will n o t b e d o c u m e n t e d in d e t a i l here. T h e cells o f P. vulgaris c o t y l e d o n s h a v e l a r g e s t a r c h g r a i n s , n u m e r o u s p r o t e i n b o d i e s m e a s u r i n g 1-10 g m in d i a m eter, a v e r y e x t e n s i v e n e t w o r k o f r o u g h E R c i s t e r n a e , a n d all o t h e r o r g a n e l l e s n o r m a l l y p r e s e n t in p h y s i o l o g i c a l l y a c t i v e cells. A n e x a m i n a t i o n o f t h i n s e c t i o n s of frozen cotyledons showed that ultracryotomy could be r e a d i l y a p p l i e d to t h e s e p l a n t o r g a n s , a n d t h a t t h e r e was e x c e l l e n t p r e s e r v a t i o n o f u l t r a s t r u c t u r e if t h e s p e c i m e n s w e r e f i x e d in a m i x t u r e o f f o r m a l d e h y d e ( 4 % , w/v), g l u t a r a l d e h y d e ( 0 . 7 5 % , v/v) a n d a c r o l e i n
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B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
Figs. 4 and 5. Ultrathin frozen sections of cotyledon of P. vulgaris labeled with rabbit anti-phaseolin IgG (0.2 mg/ml) and then with goat-anti-rabbit IgG conjugated to ferritin. Note the very dense distribution of ferritin particles over the protein bodies (PB), vesicles (V) and ER. Fig. 4: x 66,000, bar=0.5 p,m; Fig. 5: x 54,000, bar=0.5 gm
B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
423
Figs. 6 and 7. Ultrathin frozen sections of cotyledon of P. vulgaris labeled as in Figs. 4 and 5, except that the concentration of rabbit anti-phaseolin IgG was only 0.02 mg/ml. A small n u m b e r of ferritin particles are now bound, but specificity is greater here than in Fig. 4. ER cisternae are labeled, as are vesicles closely associated with Golgi apparatus (Go); mitochondria (m) are not labeled. x 100,000, b a r = 0 . 5 p,m
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B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons
(0.75%, v/v). Fixation in formaldehyde alone or in formaldehyde with acrolein resulted in poor preservation of ultrastructure and/or poor sectioning of the frozen specimens. Ultrastructural preservation of the cytoplasmic organelles in storage parenchyma cells is shown in Fig. 1. The cell contains protein bodies and a very extensive ER, as well as numerous dense vesicles. The ER cisternae are known to be covered with ribosomes (Opik 1968; Briarty 1973) and polysomes obtained from isolated ER are engaged in the biosynthesis of phaseolin (Bollini and Chrispeels 1979). Dictyosomes, i.e., Golgi apparatuses, are also clearly visible in the frozen sections (Fig. 2) although we did not observe many clearly recognizable dictyosomes consisting of a stack of cisternae with associated secretory vesicles. Mitochondria were more difficult to identify, especially at the lower magnification shown in Fig. 1, and were easily confused with small protein bodies. At higher magnification the non-uniformity in the internal structure based on the presence of cristae, made it possible to identify mitochondria (see Figs. 6, 7). Characterization of the Antibodies. Phaseolin was purified in its 18S form on sucrose gradients at pH 4.5. When subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis this pure phaseolin contained four polypeptides in the 50,000 molecularweight range (Bollini and Chrispeels 1979). No contaminants were visible on overloaded gels. The purified phasolin was used as antigen and the resulting rabbit antiserum fractionated to yield an IgG fraction. The purity of the antigen was also checked by crossed immunoelectrophoresis. A single precipitation arc was formed (Fig. 3) indicating that the antigen (phaseolin) was indeed a pure protein. The IgG fraction was used for the indirect immunolabelling experiments described below. Localization of Phaseolin. Using a high concentration of IgG (0.2 mg/ml) we observed very heavy labeling with ferritin particles over the protein bodies, the cisternae of the ER and associated ER vesicles (Figs. 4, 5); some ferritin particles could also be seen over the electron-translucent areas of cytoplasm apparently devoid of ER or proteins bodies. This low degree of specificity was considered to be a consequence of the high concentration of IgG. Using a low concentration of anti-phaseolin IgG (0.02 mg/ml) we observed lighter labeling of the same structures (protein bodies, ER cisternae and vesicles). The low concentration, on the other hand, resulted in greater specificity of the antibody-antigen reaction; the number of ferritin particles bound to the tissue section was much smaller, but they were exclusively found to be associated with protein bodies, ER cister-
nae and ER vesicles (Figs. 6, 7). In the top-right-hand corner of Fig. 7 ferritin particles are present over vesicles found in close proximity to the dictyosome (arrows) but not over other areas of the organelle. Whether this represents labeling of the dictyosome or labeling of a nearby ER cisterna is not clear. The mitochondria and the cytosol are not labeled with ferritin particles in Figs. 6 and 7, indicating the high specificity of labeling at the lowIgG concentration. Control experiments were carried out by treating section with pre-immune rabbit IgG followed by ferritinconjugated goat anti-rabbit IgG. Very few ferritin particles were visible on sections treated in this manner (data not shown), indicating that the pre-immune rabbit IgG did not bind non-specifically to the sections. The subcellular localization of phaseolin was also examined in the cotyledons of 2-d-old seedlings. At that time in cotyledon development, ferritin particles were found only over the protein bodies but not over the ER (data not shown). Such an observation is consistent with the finding that phaseolin synthesis stops towards the end of seed maturation (Sun et al. 1978; Bollini and Chrispeels 1979). Conclusion. The results presented here show that the technique of cutting ultrathin sections from frozen specimens and labeling these sections with ferritinconjugated antibodies is applicable to plant cells; ultrastructural preservation was found to be excellent and the various organelles clearly recognizable after staining. The storage protein phaseolin is localized not only in the protein bodies, but also in the cisternae and vesicles of the ER. We postulate that phaseolin is sequestered in the ER after it is synthesized by ER-bound polysomes (Bollini and Chrispeels 1979) and that the ER functions in the transport of phaseolin to the protein bodies. There is at present insufficient evidence to decide whether or not the Golgi apparatus also plays a role in this transport. Supported by grants from the National ScienceFoundation (Metabolic Biology)and North-Atlantic Treaty Organization to M.J.C., and by a grant from the U.S. Public Health Service to S.J. Singer and K.T.T.
References Bailey, C.J., Cobb, A., Boulter, D. (1970)A cotyledon slice system for the electron autoradiographic study of the synthesis and intracellular transport of seed storage protein of Vicia faba. Planta 95, 103-118 Bain, J.M., Mercer, F.V. (1966) Subcellular organisation of the developing cotyledons of Pisum sativum. Aust. J. Biol. Sci. 19, 49-67 Bollini, R., Chrispeels, M.J. (1978) Characterization and subcellu-
B. Baumgartner et al. : Reserve Protein Localization in Bean Cotyledons lar localization of vicilin and phytohemagglutinin, the two major reserve proteins of Phaseolus vulgaris L. Planta 142, 291-298 Bollini, R., Chrispeels, M.J. (1979) The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons. Planta 146, 487-50i Briarty, L.G. (1973) Stereology in seed development studies: Some preliminary work. Caryologia 25, Suppl. 289-301 Briarty, L.G., Coult, D.A., Boulter, D. (1969) Protein bodies of developing seeds of Viciafaba. J. Exp. Bot. 20, 358 372 Harris, N. (1979) Endoplasmic reticulum in developing seeds of Vicia faba. A high voltage electron microscopic study. Planta 146, 63-69 Harris, N., Boulter D. (1976) Protein body formation in cotyledons of developing cowpea (Vigna unguiculata) seeds. Ann. Bot. 40, 739 744 Kishida, Y., Olsen, B.R., Berg, R.A., Prockop, D.J. (1975) Two improved methods for preparing ferritin-protein conjugates for electron microscopy. J. Cell Biol. 64, 331 339 Laurell, C.-B. (1965) Antigen antibody crossed electrophoresis. Anal. Biochem. 10, 358 361 Nagahashi, J., Beevers, L. (t978) Subcellular localization of glycosyltransferases involved in glycoprotein biosynthesis in the cotyledons of Pisurn sativurn L. Plant Physiol. 61, 451-459
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Opik, H. (1968) Development of cotyledon cell structure in ripening Phaseolus vulgaris seeds. J. Exp. Bot. 19, 64-76 Sun, S.M., Mutschler, M.A., Bliss, F.A., Hall, T.C. (1978) Protein synthesis and accumulation in bean cotyledons during growth. Plant Physiol 61, 918-923 Ternyck, T., Avrameas, S. (1972) Polyacrylamide-protein immunoadsorbents prepared with glutaraldehyde. FEBS Lett. 23, 24-28 Tokuyasu, K.T. (1978) A study of positive staining of ultrathin frozen sections. J. Ultrastruct. Res. 63, 287-307 Tokuyasu, K.T. (i980) Adsorption staining method for ultrathin frozen sections. Proc. 38th Ann. Meet. Electr. Micr. Soc. America, pp. 760 763 Tokuyasu, K.T., Singer, S.J. (1976) Improved procedures for immunoferritin labelling of ultrathin frozen sections. J. Cell Biol. 71, 894-906 Weeke, B. (1973) Rocket immunoelectrophoresis. Crossed immunoelectrophoresis. Scand. J. Immunol. 2, Suppl. 1, 37-56
Received 23 July; accepted 22 September 1980
