Planta

Planta (1985)165:522-526

9 Springer-Verlag1985

The major albumin protein from pea (Pisum sativum L.) Localisation by immunocytochemistry N. Harris and R.R.D. Croy Department of Botany, University of Durham, South Road, Durham DH1 3LE, U.K.

Abstract. The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles. Key words: Albumin (localisation) - Cotyledon Pisum (albumin protein) - Immunocytochemistry.

Introduction During the development of Pisum sativum seeds the cotyledons accumulate carbohydrate, protein and lipid reserves in the differentiating storage parenchyma tissues (Bain and Mercer 1966). The protein fraction is divided classically into water-soluble albumins and the water-insoluble globulins (for review, see Danielson 1956). Studies of P. sativum seed proteins have been concerned mostly with the globulin fractions (Gatehouse et al. 1984) although recently the major pea albumin protein (PMA) has been purified and partially characterised (Croy et al. 1984). Purified to homogeneity, the protein contains a larger isomer (PMA-L, M r ~53000) which consists of two 25000-M r subunits and a Abbreviation: PMA =pea major albumin protein

smaller isomer (PMA-S, M r ~48000) consisting of two 24000-M~ subunits. The close homology of the isomers was demonstrated by immunological cross reaction, amino-acid composition, N-terminal amino acids, tryptic peptide mapping and CNBr-cleavage products. The total albumin fraction, which varies in amount with genetic line and method of extraction, forms between 14% and 42% of total seed protein (Grant et al. 1976; Murray 1979). It contains a few proteins which are present in amounts large enough to be considered nutritionally important. These albumin proteins have been reported to have a relatively high proportion of sulphur amino acids in comparison with the sulphur level of the globulin proteins (Davies 1976; Hurich et al. 1977; Jakubek and Przyblyska 1979; Croy et al. 1984). The albumin fraction has generally been regarded as the metabolic and enzymic protein fraction of the seeds (Boulter et al. 1967) although Murray (1979) has suggested that the albumins might be regarded as storage proteins. Unlike the globulin proteins which are localised in membranebound proteins (Graham and Gunning 1970), the major albumin fraction is not appreciably degraded during germination (Murray 1979; Tyler 1981 ; Jakubek and Przybylska ] 982) and biochemical evidence indicates that the albumin fraction, PMA is cytosolic (Croy et al. 1984). In this paper we describe the ultrastructural, immunocytochemical localisation of the PMA in storage parenchyma cells in the cotyledons of developing peas. Protein A-gold was used as the marker for binding to affinity-purified rabbit antialbumin. Previous immunocytochemical studies of protein localisation in developing seeds have concentrated on those proteins stored in protein bodies. Graham and Gunning (1970) used antibodies, with

N. Harris and R.R.D. Croy: Localisation of pea albumin protein fluorescent labelling, to show t h a t b o t h legumin a n d vicilin were localised in the s a m e p r o t e i n b o d ies in p e a cotyledons. M o r e recently B a u m g a r t n e r et al. (1980), used ferritin-labelled antibodies for the u l t r a s t r u c t u r a l localisation o f phaseolin in sections p r e p a r e d b y c r y o u l t r a m i c r o t o m y . Whilst this m e t h o d gives g o o d retention o f antigenicity, the c o n t r a s t o f u l t r a s t r u c t u r a l features was low a n d left u n r e s o l v e d the questions c o n c e r n i n g the p o t e n tial role o f the d i c t y o s o m e s in s t o r a g e - p r o t e i n t r a n s p o r t . I m m u n o c y t o c h e m i c a l localization o f storage proteins o f Vicia faba (Nieden a n d N e u m a n n 1982) was carried o u t using a Protein A - g o l d localization for vicilin a n d legumin. Tissue was fixed with f o r m a l d e h y d e a n d e m b e d d e d in glycol m e t h a c r y l a t e , a m e t h o d which resulted in r a t h e r p o o r u l t r a s t r u c t u r a l p r e s e r v a t i o n a n d quite a high level o f non-specific staining, C r a i g a n d G o o d c h i l d (1984a) h a v e used the binding o f g o a t a n t i - r a b b i t gold to sections previously labelled with r a b b i t anti-vicilin i m m u n o g l o b u l i n ( I g ) G to localise vicilin during its synthesis a n d d e p o s i t i o n in pea. I n this w o r k they labelled sections o f tissue which h a d been fixed with aldehyde a n d o s m i u m tetroxide a n d e m b e d d e d in S p u r r resin (Spurr 1969). Periodate-HC1 t r e a t m e n t o f sections was used to give a 20-fold e n h a n c e m e n t o f specific labelling b u t the final levels o f labelling o f the e n d o p l a s m i c reticul u m a n d G o l g i vesicles were still very low. C o n t r a s t was, however, better t h a n t h a t o b t a i n e d b y the c r y o u l t r a m i c r o t o m y m e t h o d o f B a u m g a r t n e r et al. (1980). V a n Driessche et al. (1981) used an unlabelled p e r o x i d a s e - a n t i p e r o x i d a s e p r o c e d u r e to localise lectin in the p r o t e i n bodies o f b o t h the cotyledons a n d the e m b r y o axis o f p e a seeds. I m m u n o c y t o c h e m i c a l m e t h o d s h a v e been used to localise the lectin o f Banhinia purpurea ( H e r m a n a n d Shann o n 1984a) a n d also c o n c a n a v a l i n A in developing jack-bean cotyledons (Herman and Shannon 1984b). T h e m e t h o d described in this p a p e r differs f r o m those described a b o v e in t h a t the tissue was fixed only in b u f f e r e d aldehyde a n d subsequently emb e d d e d in plastic w i t h o u t the use o f p r o l o n g e d heating for p o l y m e r i s a t i o n . T h e results indicate t h a t it is possible to retain a p p a r e n t l y high levels o f antigenicity whilst preserving the cellular ultrastructural features in a m a n n e r which facilitates c o n t r a s t staining a n d easy identification o f o r g a n elles.

Material and methods Pea seeds (Pisum sativum L. cv. Feltham First; Suttons Seeds, Reading, Berks., UK) were grown as described previously

523 (Evans et al. 1979), in hydroponic culture with a 16-h day at 25~ C and a night temperature of 20~ C. Material for fixation, embedding and subsequent immunocytochemical localisation studies was taken at 15 d after flowering at which point the PMA is being actively synthesised and accumulated (Tyler 1981 ; Croy et al. 1984). The albumins were extracted from mature seeds into 20 mM sodium acetate buffer pH 5.0 and the PMA purified by ammonium-sulphate fractionation, gel filtration and ion-exchange chromatography as described in Croy et al. (1984). Antibodies to the purified PMA fraction were raised in New Zealand white rabbits, the IgG fraction purified from serum (Evans et al. 1979) and specific anti-albumin antibodies isolated on affinity columns of PMA immobilised on Sepharose 4B according to the method of March et al. (1974) and Bokhout and Van Tiggele (1977). Tissue for immunocytochemical localisation was taken from cotyledons of developing seeds at 15 d after flowering. Pieces of cotyledon storage parenchyma tissue, approx. I mm 3, were fixed in a mixture of 1% glutaraldehyde and 2% formaldehyde in 50 mM Na-phosphate buffer at pH 7.0. After dehydration in a graded ethanol series the tissue was infiltrated and embedded in LR White resin (The London Resin Co., Basingstoke, Hampshire, UK). The resin was polymerized using catalyst addition although care was taken to reduce heating of the specimen by the normally exothermic reaction. Specimens were embedded in resin in aluminium foil moulds which fitted depressions in an aluminium block approx. 5.3-3 cm 3. The block, with specimens in unpolymerised resin, was cooled to -80 ~ C. After addition of the catalyst the temperature was progressively raised over a period of 6 h to room temperature. The block and specimens were maintained under N 2 during this period to prevent moisture condensation. The blocks were left overnight at room temperature before being sectioned. Thin sections were mounted on carbon-coated Formvar on 200-mesh grids. The sections were incubated for 10 min on drops of antibody (protein concentration approx. 0.2 mg ml- 1) at room temperature. After thorough washing with Na-phosphate-buffered saline pH 7.0 the grids were placed on drops of Protein A-gold colloid. Gold colloid of 20 nm diameter was made using the method of Horisberger and Rosset (1977) and coated and stabilised with Protein A (Sigma, Poole, Dorset, UK) using the procedure detailed in Roth et al. (1978). After 10 min incubation on the protein A-gold at room temperature the grids were thoroughly washed and sequentially post-stained with uranyl acetate and alkaline lead citrate. Controls included the use of pre-immune serum and the elimination of the antibody-incubation step in which eases only very occasional random labelling was observed. Stained sections were examined in an EM 400 electron microscope (Philips Industries, Eindhoven, The Netherlands) at either 60 or 80 kV.

Results and discussion T h e i m m u n o c y t o c h e m i c a l localisation o f the P M A in storage p a r e n c h y m a tissues o f developing p e a c o t y l e d o n is s h o w n in Figs. I a n d 2. D e n s e labelling o f the cytosol is a p p a r e n t in b o t h figures following p r o t e i n A - g o l d t r e a t m e n t o f sections previously i n c u b a t e d with a n t i - a l b u m i n antibodies. T h e p r o t e i n bodies which o c c u p y a large v o l u m e fraction o f the cell p r o t o p l a s t (Craig et al. 1979), were unlabelled except for the very occasional, rand o m l y distributed, gold particle. Cell walls were also generally free o f label with the exception o f

Fig. 1. Immunogold localisation of the pea major albumin protein (PMA) in cotyledon storage parenchyma cells of pea seeds harvested 15 d after flowering. Gold particles are located over the cytosolic compartment, leaving the cell wall (cw), mitochondria (rot), protein body (pb) and cisternal endoplasmic reticulum (cER) unstained, x 36000; bar = 0.5 gm Fig. 2. Immunogold staining of P M A showing a cytosolic distribution and staining associated with a plasmodesma (double-arrow), but no staining of the dictyosome cisternae (d) or the associated vesicles. Apparent labelling of ER lumen results from proximity of label to membrane (darts) or extension of particle clusters (arrow) x 42000; bar = 0.5 lam

525

N. Harris and R.R.D. Croy: Localisation of pea albumin protein

a few gold particles which were usually associated with plasmodesmata (Fig. 2, double arrow). Nuclei, amyloplasts and their starch grains, and the intercellular spaces, including the middle lamella of the wall surrounding the spaces, were unlabelled in sections except for the very occasional, randomly distributed gold particles (results not shown). After the post-staining of sections of non-osmicated tissue, most organelles appeared similar to those of conventionally osmicated tissue. The cell walls however were characteristically different in that both middle lamella and wall fibrils were not visualised in the stained sections of non-osmicated tissues. The rough, cisternal endoplasmic reticulum (ER) is mostly unlabelled although with very high adjacent levels of cytoplasmic labelling there is some apparent labelling of the ER cisternae. Careful examination of the apparent labelling of the ER lumen indicates that the gold particles are usually found at, or very close to, the ER membrane (Fig. 2, darts) or are extensions of small clusters of gold particles (Figure 2, arrow). Similarly, with the high level of cytosolic labelling the mitochondria show only a very low level of staining which is associated with the periphery of the organelles (Fig. 1). The dictyosome cisternal elements, with their peripheral reticulum and vesicles, are also unlabelled (Fig. 2). No appreciable labelling was found associated with either the apparently 'empty' or electron-opaque vesicles which are associated with the transport of the major pea vacuolar proteins (Craig and Goodchild 1984b; Harris 1984; Chrispeels 1985). Controls, including the use of pre-immune serum in place of anti-albumin and treatment of sections with Protein A-gold only, resulted in very low levels of a random distribution of gold particles over sections and grid support films (results not shown). The immunocytochemical labelling results clearly show that the major albumin fraction of developing peas is located within the cytosol rather than deposited with the major storage protein globulins, vicilin, convicilin and legumin, which are located within the membrane-bound vacuolar protein bodies. These results support the findings of Croy et al. (1984) that the PMA is cytosolic and does not therefore have a primary storage-protein function. The results do not, however, elucidate any function for this protein. Unlike the immunocytochemical studies of vicilin distribution by Craig and Goodchild (1984a) we did not have problems with low levels of specif-

ic staining and have not needed to pretreat sections with periodate-HC1. This may be attributed to either the omission of osmimn from the fixation protocols or the differences in resin-embedding techniques. The omission of osmium as a secondary fixative appears to have little effect on the general ultrastructural preservation of the tissue and sections appear to have adequate contrast by use of the conventional heavy-metal staining procedures. The possible structure-function relationships, or artefact nature, of a cytomatrix with a 'microtrabecular lattice' have been the subject of much recent interest and discussion (Porter 1984 and references therein). The immunocytochemical localisation of the major albumins gave no indication of specific distributions within the cytoplasm although the total albumin fraction is considered to contain the metabolic (or antimetabolic) and enzymic fractions of the cell proteins and might be expected to be associated with the microtrabecular lattice. We should like to acknowledge the assistance of Mr. R.A.K. Ragab in the preparation of the anti-albumin antibodies.

References Bain, J.MK., Mercer, F.V. (1966) Subcellular organisation of the developing cotyledons of Pisum sativum L. Aust. J. Biol. Sci. 19, 49-67 Baumgartner, B., Tokuyasu, K.T., Chrispeels, M.J. (1980) Immunocytochemical localisation of reserve proteins in the endoplasmic reticulum of developing beans (Phaseolus vulgaris) cotyledons. Planta i50, 419-425 Bokhout, B.A,, Van Tiggele, L.T. (1977) Complete automation of affinity chromatography. Isolation of specific antibodies. Science Tools 24, 56-57 Boulter, D., Thurman, D.A., Derbyshire, E. (1967) A disc electrophoretic study of globulin proteins of legume seeds with reference to their systematics. New Phytol. 66, 27-33 Chrispeels, M.J. (1985) The role of the Golgi apparatus in the transport and post-translational modification of vacuolar (protein body) proteins. In: Oxford surveys of plant molecular and cell biology 2, Miflin, B.J., ed. Clarendon Press, Oxford Craig, S., Goodchild, D.J., Hardham, A.R. (1979) Structural aspects of protein accumulation in developing pea cotyledons with emphasis on qualitative and quantitative changes in parenchyma cell vacuoles. Aust. J. Plant Physiol. 6, 81-98 Craig, S., Goodchild, D.J. (1984a) Periodate-Acid treatment of sections permits on grid immunogold localization of pea seed vicilin in ER and Go/gi. Protoplasma 122, 35-44 Craig, S., Goodchild, D.J. (1984b) Golgi-mediated vicilin accumulation in pea cotyledon cells is re-directed by monensin and nigericin. Protoplasma 122, 91-97 Croy, R.R.D., Hoque, M.S., Gatehouse, J.A., Boulter, D. (1984) The major albumin proteins from pea (Pisum sativum L). Purification and some properties Biochem. J. 218, 795-803 Danielson, C.E. (1956) Plant proteins. Annu. Rev. Plant Physiol. 7, 215-236

526 Davies, D.R. (1976) Variations in the storage proteins of peas in relation to sulphur amino acid content. Euphytica 25, 717-724 Evans, I.M., Croy, R.R.D., Hutchinson, P., Boulter, D., Payne, P.I., Gordon, M.E. (1979) Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.) Planta 144, 455-462 Gatehouse, J.A., Croy, R.R.D., Boulter, D. (1984) The synthesis and structure of pea storage proteins. CRC Crit. Rev. Plant Sci. 1,217-314 Graham, T.A., Gunning, B.E.S. (1970) The localization of legumin and vicilin in bean cotyledon cells using fluorescent antibodies. Nature 228, 81-82 Grant, D.R., Sumnar, A.K., Johnson, J. (1976) An investigation of pea seed albumins. Can. Inst. Food Sci. Technol. J. 9, 84-91 Harris, N. (1984) Immunocytochemical localization of the intra cellular secretion of major legume seed storage proteins via the Golgi apparatus. (Abstr.) Proc. R. Microsc. Soc. 19, S16 Herman, E.H., Shannon, L. (1984a) lmmunocytochemical evidence for the involvement of the Golgi Apparatus in the deposition of seed lectin of Bauhinia parparea (leguminosae) Protoplasma 121, 163-170 Herman, E.M., Shannon, L. (1984b) Immunocytochemical localization of concanavalin A in developing Jack bean cotyledons. Planta 161, 97-104 Horisberger, M., Rosset, J. (1977) Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochem. Cytocbem. 25, 295-305 Hurich, J., Parsysz, H., Przybylska, J. (1977) Comparative

N. Harris and R.R.D. Croy: Localisation of pea albumin protein study of seed proteins in the genus Pisum. II Amino acid composition of different protein fractious. Genet. Pol. 18, 241-252 Jakubek, M., Przybylska, J. (1979) Comparative study of seed proteins: the genus Pisum. Electropboretic patterns and amino acid composition of albumin fraction separated by gel filtration. Genet. Pol. 20, 369-380 Jakubek, M., Przybylska, J. (1982) Changes of electrophoretic albumin patterns in germinating seed of five different Pisurn lines. Pisum Genet. Assoc. Pisum Newsl. 14, 26-28 March, S.C., Parikh, I., Cuatrecasas, P. (1974) A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 60, 149-152 Murray, D.R. (1979) A storage role for albumin in pea cotyledons. Plant Cell Environ. 2, 221-226 Nieden, U., Neumann, D. (1982) Electron microscope immunocytochemical localization of storage proteins in Vieia faba seeds. Eur. J. Cell Biol. 26, 228-233 Porter, K.R. (1984) The cytomatrix: a short history of its study. J. Cell Biol. 99, 35-125 Roth, J., Bendayan, M., Orci, L. (1978)Ultrastructural localisation of intracellular antigens by the use of protein-A gold complex. J. Histochem. Cytochem. 26, 1074-1081 Spurr, A.R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. Ultrastruct. Res. 26, 31-43 Tyler, M. (1981) Ph.D. Thesis, University of Durham Van Driessche, E., Smets, G., Dejaegere, R., Kanarek, L. (1981) The immuno-histochemical localization of lectin in pea seeds (Pisum sativum L.) Planta 153, 287-296 Received 21 December 1984; accepted 26 February 1985