Protoplasma 105, 333--339 (1981) PROTOPLASMA �9 by Springer-Verlag 1981

Pea Seed Storage Proteins - - Immunocytochemical Localization

with Protein A-Gold by Electron Microscopy

Brief Report

STUART CRAIG ':" and ADELE MILLERD

CSIRO, Division of Plant Industry, Canberra

Received July 22, 1980 Accepted in revised form October 24, 1980

Summary The pea seed storage proteins legumin and vicilin have been localized by electron microscopy using a post-embedding immunocytochemical double-labelling technique. Highly purified antibodies from sheep, specific to legumin and vicilin, were sequentially reacted on sections of pea cotyledon tissue embedded in glycol methacrylate or Spurr's epoxy resin. The sheep antibodies were visualized indirectly by reaction with Staphylococcal Protein A labelled with colloidal gold. Two discrete sizes of colloidal gold particles can be used for simultaneous localization of two antigens. In near mature tissue, the storage proteins are restricted to organelles, called protein bodies. Individual protein bodies contain both legumin and vicilin.

Keywords: Immunocytochemistry; Pea seed; Protein histochemistry; Storage proteins.

1. Introduction

We recently demonstrated the localization, by immunofluorescence, of legumin and vicilin 4 (THOMSON et al. 1978), the storage proteins of pea seeds, in sections of tissue embedded in glycol methacrylate (GMA) (CRAIG et al. 1979, 1980). However important questions such as the cytoplasmic origin and mode of transport of the proteins into the storage vacuoles remain unanswerable at the resolution of the light microscope, and the joint sequestration of both Iegumin and vicilin in all protein bodies could not be established. Protein A, a cell wall protein from Staphylococcus aureus, has high affinity for the Fc fragment of immunoglobulin G (IgG) molecules from a variety of species (GouDSWAARD et al. 1978). Numerous marker molecules such as ferritin, peroxidase, and colloidal gold (RoTtI and BINDER 1978) have been coupled to protein A and used successfully in immunocytochemical studies

* Correspondence and Reprints: CSIRO, Division of Plant Industry, P.O. Box 1600, Canberra City, A.C.T. 2601, Australia.

0033-183X/81/0105/0333/$ 01.40

334 S. CRAIG and ADULt MILLERD

of cell surface antigens. Recently ROTH et al. (1978), BATTEN and HOVKINS (1979), and BENDAYAN et al. (1980) have employed protein A-gold complexes to visualize antigen-antibody complexes on ultrathin sections of epoxy embedded tissues. To date, these techniques have not been utilized on higher plant tissues. We report here the application of protein A-gold complexes to localize anti- bodies bound specifically to legumin and vicilin 4 in ultrathin sections of plastic-embedded pea cotyledon tissue.

2, M a t e r i a l s and M e t h o d s Plants of Pisum sat ivum (PI/G 086 a selection from cv. Greenfeast) were grown under con- ditions of controlled environment as described elsewhere (MILLeRD and SPENCER 1974). Tissue Preparation. Cotyledon tissue was fixed in 1 to 3~ glutaraldehyde in 25 mM phosphate buffer pH 7.1 for 1.5 hours, rinsed in buffer and dehydrated through ethanol before embedment in epoxy resin (SPuRR 1969). For GMA embedment, tissues were de- hydrated through a graded GMA/H.20 series. Our original GMA mixture (CRAIG et al. 1979) would not yield sufficiently thin sections to be useful for electron microscopy, and accordingly more benzoyt peroxide (1.5~ was added. The GMA was polymerized under UV light, as described previously. All preparative steps were carried out at 20 ~ Preparation o] Protein A-gold Complex. Preparation of the protein A-gold complex was adapted from ROTH et aI. (1978). Colloidal gold, mean diameter 25 nm (gold-25) or 46 nm (gold-46), was prepared by reducing 0.01~ tetrachloroauric acid (Merck, Darmstadt, F.R.G.) with sodium citrate (FI~ENS 1973). The minimal amount of protein A (Pharmacia, Uppsala, Sweden) for full stabilization of the colloid was determined (RoTH et aI. 1978), and a 200/0 excess (RoTtt et al. 1978) used in the preparation of the protein A-gold complex. Protein A-gold 25 (10 ml) was centrifuged at 100,000 • g for 30 minutes and the sedimented protein A-gold complex resuspended in 4 ml phosphate buffered saline pH 7.4 (PBS) containing 0.02~ polyethylene glycol (PEG, Carbowax 20 M, Union Carbide Co.) and 0.020/0 azide (RoTH et al. 1978). This preparation was diluted • 10 with PBS-azide before use. Protein A-gold 46 (10 ml) was centrifuged at 23,000 • g for 15 minutes, the sedimented complex was resuspended in 10 ml PBS containing 0.02~ PEG, and recentrifuged at 23,000 X g for 15 minutes. The sedimented complex was resuspended in 2 ml PBS-azide containing 0.02~ PEG, and a small amount of material which did not resuspend was discarded. This pre- paration was used undiluted. Irnmunocytochemical Labelling. Anti-legumin and anti-vicilin 4 were purified by affinity chromatography, as described previously (CRAIG et al, 1979). Sections were collected on parlodion coated copper grids and incubated on drops of antibody in PBS-azide in the following sequence: 1. sheep anti-legumin, 20 p.g/ml; 2. rabbit anti-sheep 1 0.2-0.4mg/ml; 3. protein A-gold 25; 4. sheep anti-vicilin 4, 20 ug/ml; 5. rabbit anti-sheep, 0.2-0.4 mg/ml; 6. protein A-gold 46. For labelling a single antigen the appropriate antibody was followed by steps 2 and 3. Each incubation was carried out for 10 minutes at 20 ~ and was followed by three 1 ml washes in PBS-azide from a Pasteur pipette. Grids were finally rinsed in distilled water and dried. GMA sections were examined without heavy metal counterstaining in either a Philips EM 200 or JEOL 100 CX electron microscope at 100 kV or 80 kV respectively, and in both instruments a liquid nitrogen cooled anticontamination trap was operated; there was no apparent boiling of the methacrylate under these conditions. Epoxy sections were counter- stained with saturated uranyl acetate in 50~ ethanol.

The IgG fraction of rabbit antiserum raised against sheep IgG (ICN Pharmaceuticals).

Pea Seed Storage Proteins 335

Irnrnunocytochemical Controls. The following immunocytochemical controls have been em- ployed to establish the validity of our observations for single antigens. a) Substitution of the antibody in step 1, i.e., sheep anti-legumin or sheep anti-vicilin 4 (both IgGi), with sheep anti-ovalbumin (IgGi). b) Factorial dilution of the sheep anti-legumin, sheep anti-vicilin 4 and the sheep anti- ovalbumin antibodies with PBS prior to their use in step 1. c) Absorption of the step 1 antibodies with an excess of antigen from a PBS extract of protein bodies, prior to their use in step 1. d) Use of sheep IgG_~ (isolated as described by BRANnTZA~G 1973) in step 1. e) Omission of step 1, the primary antibody. f) Treatment of sections with protein A-gold (step 3) only. When detecting both legumin and vicilin 4, the controls were: g) Absorption of the antibodies with a PBS extract of protein bodies, prior to their use in steps 1 and 4. h) Labelling in the sequence, steps 1, 2, 3, and 6 to verify that the protein A-gold 46 does not exchange with protein A-gold 25 or fill unsaturated sites on the rabbit antibodies used in step 2.

3. Resul t s

Results of the control reactions are shown only for control (g) (Fig. 3 see be- low). Control reaction (a) resulted in deposition of gold over the protein bodies, but in a dilution-dependent manner. Dilution of the anti-ovalbumin 100 fold (control b) resulted in negligible binding of gold, whereas similar dilution of the anti-legumin or anti-vicilin 4 resulted in only a small decrease in the gold bound. Controls (c), (d), (e), and (f) resulted in negligible gold bound to the sections. Control (h) showed negligible gold-46, indicating that the second protein A gold probe had not exchanged with the protein A gold 25 or detected unsaturated rabbit IgG sites from step 2. Also, we have varied the reaction time of steps 1, 2, and 3 in the labelling and find no increase in bound gold (by subjective assessment, not by counting particles) with time over 10 minutes. In fact most sites are saturated within 3-5 minutes. Labelling of epoxy sections for legumin or vicilin 4 results in similar binding of gold to all protein bodies, and is shown for vicilin 4 in Fig. 1. The distribution of gold over the cytoplasm is uniformly low and is not different from the background labelling [controls (e) and (f) not illustrated]. Figs. 2 and 3 show respectively, treated and control micrographs of GMA sections on which both legumin and vicilin 4 have been localized. In Fig. 2, 25 and 46 nm gold particles bound to protein A have been used to visualize legumin and vicilin 4 respectively. All protein bodies contain both small and large gold particles, showing the cohabitation of legumin and vicilin 4. In the control micrograph (Fig. 3), the anti-legumin and anti-vicilin 4 were replaced by antibodies that had been adsorbed with an extract of protein bodies. Negligible numbers of gold particles, small or large, have bound. Particle diameters have been measured from Fig. 2 and their frequencies

336 S. CRAIG and ADELE MILLERD

Fig. 1. Section of pea cotyledon tissue (18 days after flowering) embedded in Spurr's epoxy resin and labelled for vicilin 4 with 25 nm gold particles. Most of the particles are single but some aggregates are present (see inset). P protein body, PI plastid

Figs. 2 and 3. Sections of GMA embedded cotyledon tissue, 20 days after flowering. Fig. 2 labelled for legumin with 25 nm gold particles and for vicilin 4 with 46 nm gold particles. Note the difference between the latter (inset) and the aggregated 25 nm particles seen in places in Fig. 1. Fig. 3 section showing low level of non-specific labelling given in control (g). P protein body

Pea Seed Storage Proteins 337

plotted (Fig. 4). Two distinct peaks, at 25-27 and 46-47 nm diameter, are apparent, and these are in close agreement with similar measurements made independently on the small and large protein A-gold preparations. There are always more small gold particles than there are large particles. If protein A-gold 25 is used to visualize both legumin and vicilin 4 (not illustrated) many more gold particles decorate the protein bodies than when golds 25 and 46 are used, as in Fig. 2. We therefore do not believe that ratio of small to large gold reflects the relative amounts of legumin and vicilin 4. Apparently protein A-gold 46 binds less efficiently than does protein A-gold 25. Analysis of Fig. 2 reveals that 980/0 of the gold is bound to protein

100

9C

8C

-5 7C ~ 6c ~- 5o ~ 40 ~ so

2C 10

19 23 27 31 35 39 43 47 51 55

Diameter of gold particle (nm)

Fig. 4. Size distribution of gold particles (n = 421) measured from Fig. 2. Distinct peaks at 25-27 and 46-47 nm are apparent

bodies, which account for 730/0 of the area analyzed. This represents 14 par- ticles (large and small) per ~tm 2 of exposed protein body, compared with 0.7 particles per ~m 2 of cytoplasm.

4. D i s c u s s i o n

Protein A coupled to colloidal gold has been used to visualize the pea seed storage proteins legumin and vicilin 4 in sections of plastic-embedded tissue. Sequential labelling on a single section for legumin and vicilin 4 with protein A bound to 25 and 46 nm gold particles respectively has established unequi- vocally that both proteins are sequestered within the same protein body (for discussion of previous literature on this point see CRAIG et al. 1980). In conjunction with the high resolution of the electron microscope, the procedure described here should permit a detailed investigation of the synthesis and sequestration of the pea seed proteins, not previously possible using immuno- fluorescence. Preliminary observations of younger tissue suggest that both legumin and vicilin 4 can be detected in the cytoplasm. The immunolocalization technique employed in the current report can be successfully applied both to GMA sections, and, as previously described by

338 S. CRAIG and ADELE MILLERD

BATTEN and HOPKINS (1979) and BENDAYAN et aL (1980), to epoxy sections. Subjective comparison of the gold labelling density reported here (Fig. 1) with the fluorescent labelled image described earlier (Fig. 1 in CRAIG et al.

1979) suggests that significantly less marker binds per protein body in the method reported here. This apparent discrepancy has been observed re- peatedly, and the reasons for it are unclear. But, glutaraldehyde fixation was used in the present study, in contrast to the formaldehyde fixation used pre- viously, and, as ROTH et al. (1978) detailed, protein A may bind up to 4 Fc fragments. This would produce a gold to antigen ratio less than the com- parable ratio with the fluorescent-labelled antibody technique, which amplifies the antigen (STERNBEROER 1979). We have regularly observed a greater density of bound gold on GMA sections relative to epoxy sections, and presumably this reflects an increased loss of antigenicity during processing and embedment in epoxy resin. Etching epoxy sections with 10~ hydrogen peroxide prior to immunolabelling does not increase the number of accessible antigenic sites (CRAIG, unpublished). Of the sheep G immunoglobulins, Staphylococcal protein A recognizes only subclass 2 (GouDSWAARD et al. 1978). Since sheep anti-legumin and anti- vicilin 4 are in subclass 1 of the IgG fraction (CRAIG et al. 1980), it was necessary to introduce an intermediate, in our case the IgG fraction of rabbit antibodies raised against sheep IgG, which is readily recognized by protein A. Such an indirect technique may increase the sensitivity of the method (STERN- BEROER 1979). In verifying the specificity of an anti-legumin and anti-vicilin (both IgGi) using the ELISA technique (CRAIG et al. 1980) we used sheep anti-ovalbumin (also IgGi) as the control antiserum. But when sheep anti- ovalbumin is used as the primary antiserum (step 1) in the present system, there is a pseudo-specific deposition of gold over the protein bodies. Similar false or apparent immunospecificity has been recently described by GRUBE (1980), GRUBE and WEBER (1980) and JULLIARD et al. (1980) and has been ascribed respectively to ionic interaction between IgG molecules and unknown components in the tissue, and chance homologies in the amino acid sequences of two otherwise unrelated proteins. The visualization of antibodies bound to a section by a protein A-gold com- plex is rapid, simple and economical in its consumption of antiserum, pro- tein A, and gold. Most importantly, in contrast to ferritin-labelled antibodies (PARR 1979; CRAm, unpublished) non-specific adsorption of gold to the tissue is negligible.

Acknowled[~ements

Technical assistance from Mrs. CELIA MILLER and Miss J. FLANIGAN is gratefuliy acknowi- edged. Drs. W. F. DUDMaN, R. A. DAVEY, and D. J. GOODCHILD have assisted in discussions on the immunology.

Pea Seed Storage Proteins 339

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