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Stratification of Storage Proteins in the Protein Storage Vacuole of Developing Cotyledons of Pisum sativum L.
j. Plant Physiol. Vol. 145. pp. 437-442 (1995)
Stratification of Storage Proteins in the Protein Storage Vacuole of Developing Cotyledons of Pisum sativum L. GISELBERT HINZ, BIRGIT HOH, INGE HOHL,
and DAVID
G. ROBINSON
Pflanzenphysiologisches Institut der Universitat Gottingen, Untere Karspiile 2, D-37073 Gottingen, Germany Received June 15, 1994 . Accepted September 5, 1994
Summary
Legumin, vicilin and pea albumin are the most prominent storage proteins in pea seeds. In order to localize these proteins in the developing cotyledons we have employed an improved fixation procedure for immunogold labelling for electron microscopy. Profiles of protein bodies (PB, 1-2 Ilm diameter) label heavily and specifically with antibodies against vicilin and legumin. By contrast, such PB are only weakly labelled with antibodies against pea albumin 1. Antibodies against pea albumin 1 label a narrow, granulated zone at the luminal borders of the deposits. Depending on their size, storage protein deposits at the tonoplast of the protein storage vacuole (PSV) show different labelling patterns: small deposits label positively only with vicilin antibodies; labelling with legumin is seen in large deposits but is restricted to the tonoplast rather than luminal borders of the deposits. We frequently observed profiles of storage protein deposits at the tonoplast of the PSV, which were elongated and protruded into the cytoplasm. A gradient of legumin labelling in these deposits was seen. We interpret such images as stages in the formation of PB, which apparently arise by budding at the surface of the PSV.
Key words: Legumin, pea albumin, protein body, protein deposition, stratification, vicilin. Abbreviations: BSA-C = acetylated BSA; PA 1 = pea albumin 1; PB = protein body(ies); PSV = protein storage vacuole(s); TBS = tris buffered saline. Introduction
Plants, unlike animals, can store proteins as part of a strategy to provide the plant with sufficient reserves of amino acids during seed germination or at the begin of the growing season in the case of perennial woody plants. These proteins are usually found in membrane-bound vesicles termed protein bodies (PB), whose size, morphology and origin in the cell is not uniform (Pernollet, 1978; Lott, 1980). In many cereals the PB arises by direct vesiculation of the rough endoplasmic reticulum, e.g. in maize (Larkins and Hurkman, 1978) and rice (Bechtel and Juliano, 1980) endosperms. By contrast, in the cotyledons of legumes PB are formed from the protein storage vacuole (PSV), which according to some authors is a transformed vegetative vacuole, and to others has developed de novo out of the endoplasmic reticulum (see Robinson et al., 1995 for a discussion of these two possibilities ). © 1995 by Gustav Fischer Verlag, Stuttgart
The proteins stored in PB differ considerably in their structure and properties. As a general rule it is the water and salt insoluble but alcohol or acid soluble prolamins and glutelins that constitute the PB of cereals (Shewry and Tatham, 1990), and the water soluble albumins and salt soluble globulins (Shotwell and Larkins, 1988) that are characteristic of the legume PB. Normally the content of cereal and legume PB is electron opaque and stains homogenously, with only few exceptions, e.g. the maize PB (Lending and Larkins, 1989). However, the synthesis of storage proteins is known to be developmentally regulated with some polypeptides being synthesized and deposited in the PB before others (Higgins, 1984). This is particularly well exemplified in the maturing pea cotyledon, where it has been established that the synthesis of vicilin proceeds that of pea lectin, convicilin and legumin (Chrispeels et al., 1982; Gatehouse et al., 1982; Higgins et al., 1983; Millerd et al., 1978; Spencer et al., 1980; Wenzel et al., 1993). One might therefore ask whether or
438
GISELBERT HINZ, BIRGIT HOH, INGE HOHL,
and DAVID G.
ROBINSON
a
PS¥
c Fig. 1: Immunogold detection of vicilin (a), legumin (b), and pea albumin (c, d) in storage protein deposits held within protein storage vacuoles (PSV) or protein bodies (PB). Arrowheads in (c) indicate a peripheral distribution of granular substances that colocalize with the PA-l antigen. (a) x 43.500; (b) x 35.500; (c) x 28.000; (d) x 35.000; bars throughout = 0.5 Jlm.
Protein stratification
439
.,
~
... _ - - - -
a
.
~I
;""1
i
~.
:;
Fig. 2: Immunogold detection of vicilin (a) and legumin (b) in young PSV. Arrows point to clumps of storage protein at the periphery of the PSV. The larger arrows in (b) indicates that, in contrast to the other clumps, the deposition of legumin in this clump has started. In (a) the PSV is surrounded by a vicilin-positive cisternal compartment similar to those previously described by Craig and Goodchild (1984). (a) x 38.750; (b) x 39.200; bars = 0.5 J!m.
440
GISELBERT HINZ, BIRGIT HOH, INGE HOHL, and DAVID G. ROBINSON
not the various storage polypeptides are stratified into concentric zones in the pea cotyledon PB. Using immunogold labelling we provide here an answer to this question. Material and Methods
Plant material As previously given (Hoh et al., 1995; Robinson et aI., 1995).
Immunocytochemistry One mm thick slices of cotyledon tissue were left to stand for 12-16h at 4°C in primary fixative (2 % w:v paraformaldehyde + 1 % v: v glutaraldehyde in 50 mM cacodylate buffer pH 7.4). After washing 3 x 10 min in cold cacodylate buffer the tissue was immersed in secondary fixative (1 % w:v OS04 + O.S% w:v potassium hexaferrocyanate in 50 mM cacodylate buffer, pH 7.4) for 2 h at room temperature. The tissue was then washed 2 x 10 min in cacodylate buffer and 2 x 10 min in H 20 before removing excess aldehyde through a borohydride (0.5% w:v KBH4; 30 min, RT) treatment. After dehydrating in ethanol the tissue was embedded in London Resin White (hard grade), using heat polymerisation (60°C; 20 - 24 h). Thin sections were picked up on formvar-coated nickel grids and then floated on blocking solution (3 % BSA + 0.2 % BSA-C in TBS) for 30 min before exposing to the primary antibody solution for 1 h at room temperature. After washing (4 x 5 min in 1 % BSA + 0.07 % BSA-C in TBS) the sections were incubated for 1 h in 10nm gold conjugated secondary antibody solution (diluted 1: 30 in 1 % BSA, 0.07 % BSA-C in TBS). The grids were washed again (3 x 5 min in 1 % BSA, 0.07 % BSA-C in TBS; 2 x 5 min in H 20; 10 min in 1 % glutaraldehyde; 3 x 5 min in H 20) before poststaining in uranyl acetate and lead citrate. Sections were observed in a Philips CM 10 electron microscope operating at sokV.
Primary antibodies and their dilution Polyclonal antibodies against three different storage polypeptides from pea cotyledons were employed: - affinity purified antibodies raised in rabbits against legumin (Casey, 1979), diluted 1: 100-200 in 1 % BSA + 0.07% BSA-C in TBS. - affinity purified antibodies raised in rabbits against vicilin (Croy et aI., 19S0), diluted 1:4 in 1 vol 1 % BSA + 0.07% BSA-C in TBS; 2 vol fresh pea leaf extract (Melroy and Herman, 1991). - affinity purified antibodies raised in goats against the sulfur-containing albumin PA 1 (Higgins et aI., 1986), diluted 1: 4 in 1 vol low fat milk; 2 vol fresh pea leaf extract.
Reagents for immunocytochemistry Acetylated BSA (BSA-C) was purchased from Aurion, Biotrend (Cologne, Germany). Gold coupled secondary antibody conjugates (GARG, RAGG) were obtained from Biocell (Cardiff, UK). Grids and LR-White were from Plano (Marburg, Germany). Results
Immunolocalization of vicilin and legumin In thin sections of pea cotyledon cells it is easy to recognize storage protein deposits because of their electron dense character. A positive and very dense labelling with gold coupled vicilin antibodies was found for PB and for PSV in all stages of development (Figs. 1 a, 2 a). By contrast, whereas PB were also heavily labelled by legumin antibody-gold conjugates (Fig. 1 b) small deposits of storage proteins at the periphery of young PSV were free of gold particles (Fig. 2 b). Larger clumps of storage proteins in older PSV did, however, show
Fig. 3: Immunogold localization of legumin in large storage protein deposits at the periphery of PSV. Gradients of legumin incorporation are indicated by arrows. (a) Median section through a protruding clump of storage proteins; x 34.500. (b) Tangential sections through protein clumps indicating the blebbing of PB, x 40.600. Bars = 0.5/lm.
Protein stratification
441
CYTOSOL
TP~_ PSV
Fig.4: Schematic representation of the events leading to the production of a protein body (PB) in pea cotyledons. The temporal difference in vicilin (first) and legumin (second) depositions is indicated accordingly.
!
VICILIN
a positive labelling for legumin. This was restricted to that part of the deposit adjacent to the tonoplast rather than the regions facing the lumen of the PSV (Figs. 1 b, 2 b). In very large storage protein clumps, which protruded into the cytoplasm, there was a clear gradient in immunogold labelling with legumin antibodies (Fig. 3 a). Profiles of such clumps were frequently obtained, which gave the impression that PB arise by budding of storage protein clumps from the periphery of the PSV (Fig. 3 b).
Immunolocalization ofpea albumin 1 (PA 1) In a previous report Craig (1986) described that PA 1 is restricted to the outermost layers of storage protein deposits, which are found only in cells of the embryonic axis of pea embryos. We confirm here this peculiar labelling pattern (Fig. 1 c), but have seen that it also occurs in some, but not all, of the cotyledon cells. It also only seems to be present when the periphery of such protein deposits has a granular substructure (Fig. 1 c). When this is not visible anti-PA 1 immunogold labelling is randomly distributed in the storage protein depositis at the surface of the PSV, albeit at lower densities than with anti-vicilin or anti-legumin. This is to be expected since PA 1 is a much less abundant storage protein. PB at this stage show only very little labelling (Fig. 1 d). This we interpret as reflecting the fact that albumin is synthesized late in pea cotyledon maturation and PB that have already budded off will therefore not contain albumin.
Discussion
The major storage proteins in peas are the 11 S legumin and 7S vicilin globulins (Derbyshire et ai., 1976). In addition there are smaller amounts of albumins (Croy et al., 1984), and lectin (Higgins et ai., 1983). With the exception of some albumins, which have no signal peptide and remain in the cytosol (Harris and Croy, 1986; Higgins et ai., 1987), all of these polypeptides are synthesized at the rough endoplasmic reticulum where they enter the en do membrane system co-
~
LEGUMIN
translationally (Miintz, 1989), and subsequently accumulate in PB (Pernollet, 1978; Lott, 1980). The kinetics of mRNA transcription and translation in relation to storage protein formation have been followed on numerous occasions by several groups (Higgins, 1984; Wenzel et ai., 1993). Agreement exists as to the following temporal order for storage protein synthesis: vicilin - lectin - convicilin - legumin - albumin. Since there is no evidence for more than one type of PB in maturing pea cotyledons one might expect the individual polypeptide types to accumulate in the PB in sequential, zonal manner. Our results indicate that this is to a certain extent true for the major storage proteins vicilin and legumin. A parallel situation is encountered in the mature PB of maize endosperm where the lightly staining central core is comprised of a-zeins and the darkly staining outer shell has principally (3- and 'Y-zeins (Lending and Larkins, 1989). A comparison of young (lying in outermost endosperm layers) and old (lying in more centrally located cells) PB in maize suggested the a-zeins penetrate the (3- and 'Y-zeins, which are synthesized earlier, and in young PB make up the uniform darkly staining content. However, in contrast to the maize zeins, vicilin and legumin intermix, the mature PB in pea cotyledons giving no indication of a sequential accumulation of storage proteins. Our results also indicate that at least some of the PB arise from the PSV by budding or blebbing (schematically presented in Fig. 4). The contrasts with the idea that the PSV gradually accumulates storage proteins and then fragments or subdivides into large numbers of PB (see Robinson et ai., 1995, for a discussion). The latter concept has been made especially attractive because it provides an explanation to the problem of dealing with excess membrane in the PSV delivered by vesicle-mediated storage protein transport (Craig et ai., 1979). Our model for PB formation is, in this respect, equally compatible with the need to accomodate a flow of membrane to the PSV. However, as we have shown the (macro) vesiculation event leading to the formation of a PB in pea cotyledons is associated by a differential deposition of storage polypeptides. Two questions arise as a consequence of this alternative hypothesis. Firstly, why do storage proteins aggregate in clumps more or less regularly spaced along
442
GISELBERT HINZ, BIRGIT HOH, INGE HOHL, and DAVID G. ROBINSON
the tonoplast of the PSV? Secondly, do these proteins accumulate at these loci from within the PSV or are they the result of a specific fusion of storage protein-containing vesicles at predetermined sites? Acknowledgements
We gratefully acknowledge the receipt of antisera from Drs. R. Casey (Norwich, UK), R. R. D. Croy (Durham, UK) and T. J. V. Higgins (Canberra, Australia). We thank Sibille Hourticolon for photographic work and Heike Freundt for help in typing the manuscript. Bernd Rauffeisen prepared Fig. 4. This work was supported by funds from the Deutsche Forschungsgemeinschaft.
References BECHTEL, D. B. and B. O. JULIANO: Formation of protein bodies in the starchy endosperm of rice (Oryza sativa L.): a reinvestigation. Ann. Bot. 45,503-509 (1980). CASEY, R. D.: Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds. Biochem. J. 177, 509-520 (1979). CHRISPEELS, M. T., T. J. V. HIGGINS, and D. SPENCER: Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons. J. Cell Biol. 93, 306-313 (1982). CRAIG, S.: Compartmentation of albumin and globulin storage proteins in protein deposits of pea embryo axis cells. Protoplasma 132, 107 -109 (1986). CRAIG, S. and D. J. GOODCHILD: Periodate-acid treatment of sections permits on-grid immunolocalization of pea seed vicilin in ER and Golgi. Protoplasma 122, 35-44 (1984). CRAIG, S., D. J. GOODCHILD, and A. R. HARDHAM: Structural aspects of protein accumulation in developing pea cotyledons. I. Qualitative and quantitative changes in parenchyma cell vacuoles. Austr. J. Plant Physiol. 6, 81-98 (1979). CROY, R. R. D., J. A. GATEHOUSE, I. M. EVANS, and D. BOULTER: Characterization of the storage protein subunits synthesized in vitro by polyribosomes and RNA from developing pea (Pisum sativum L.). II. Vicilin. Planta 148, 57 -63 (1980). CROY, R. R. D., M. S. HOQUE, J. A. GATEHOUSE, and D. BOULTER: The major albumin proteins from pea (Pisum sativum). Purification and some properties. Biochem.J. 218, 795-803 (1984). DERBYSHIRE, E., D. J. WRIGHT, and D. BOULTER: Legumin and vicilin, storage proteins of legume seeds. Phytochem. 15, 3 - 24 (1976). GATEHOUSE, J. A., I. M. EVANS, D. BROWN, R. R. D. CROY, and D. BOULTER: Control of storage protein synthesis during seed development in pea (Pisum sativum L.). Biochem. J. 208, 119-127 (1982). HARRIS, N. and R. R. D. CROY: The major albumin protein from pea (Pisum sativum L.). Localisation by immunocytochemistry. Planta 165, 522-526 (1986).
HIGGINS, T. J. V.: Synthesis and regulation of major proteins in seeds. Annu. Rev. Plant Physiol. 35, 191-221 (1984). HIGGINS, T. J. V., L. R. BEACH, D. SPENCER, P. M. CHANDLER, P. M. RANDALL, R. J. BLAGROVE, A. A. KORTT, and R. E. GUTHRIE: cDNA and protein sequence of a major pea seed albumin (PA 2: Mr 26000). Plant Molec. Biol. 8, 37 -45 (1987). HIGGINS, T. J. V., P. M. CHANDLER, P. J. RANDALL, D. SPENCER, L. R. BEACH, R. J. BLAGROVE, A. A. KORTT, and A. INGLIS: Gene structure, protein structure, and regulation of a sulfur-rich protein in pea seeds. J. Biol. Chern. 261, 11124-11130 (1986). HIGGINS, T. J. V., M. J. CHRISPEELS, P. M. CHANDLER, and D. SPENCER: Intracellular sites of synthesis and processing of lectin in developing pea cotyledons. J. Biol. Chern. 258, 9550-9552 (1983). HOH, B., G. HINZ, and D. G. ROBINSON: Protein storage vacuoles form de novo during pea cotyledon development. J. Cell Sci. (in press). LARKINS, B. A. and W. J. HURKMAN: Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiol. 62, 256-263 (1978). LENDING, C. R. and B. A. LARKINS: Changes in the zein composition of protein bodies during maize endosperm development. Plant CellI, 1011-1023 (1989). LOTT, J. N. A.: Protein bodies. In: TOLBERT, N. E. (ed.): The Biochemistry of Plants 1, pp. 589-624. Academic Press, New York (1980). MELROY, D. L. and E. M. HERMAN: TIP, an integral membrane protein of the protein-storage vacuoles of the soybean cotyledon undergoes developmentally regulated membrane accumulation and removal. Planta 184, 113-122 (1991). MILLERD, A., J. A. THOMPSON, and H. E. SCHROEDER: Cotyledonary storage proteins in Pisum sativum. III. Patterns of accumulation during development. Austr. J. Plant Physiol. 5, 519-534 (1978). MUNTZ, K.: Intracellular protein sorting and the formation of protein reserves in storage tissue cells of plant seeds. Biochem. Physiol. Pflanzen 185, 315-335 (1989). PERNOLLET, J. Protein bodies of seeds: ultrastructure, biochemistry, biosynthesis and degradation. Phytochem. 17, 1473 -1480 (1978). ROBINSON, D. G., B. HOH, G. HINZ, and B.-K. JEONG: One vacuole or two vacuoles: do protein storage vacuoles arise de novo during pea cotyledon development? J. Plant Physiol. (in press). SHEWRY, P. R. and A. S. TATHAM: The prolamin storage proteins of cereal seeds: structure and evolution. Biochem. J. 267, 1-12 (1990). SHOTWELL, M. A. and B. A. LARKINS: The biochemistry and molecular biology of seed storage proteins. In: MIFLIN, B. J. (ed.): The Biochemistry of Plants, Vol. 15, pp. 297 - 345. Academic Press, New York (1988). SPENCER, D., T. J. V. HIGGINS, S. C. BUTTON, and R. A. DAVEY: Pulse-labelling studies on protein synthesis in developing pea seeds and evidence of a precursor form of legumin small subunit. Plant Physiol. 66, 510-515 (1980). WENZEL, M., H. GERs-BARLAG, A. SCHIMPL, and H. RUDIGER: Time course of lectin and storage protein biosynthesis in developing pea (Pisum sativum) seeds. Biol. Chern. Hoppe-Seyler 374, 887894 (1993).
c.:
Stratification of Storage Proteins in the Protein Storage Vacuole of Developing Cotyledons of Pisum sativum L. GISELBERT HINZ, BIRGIT HOH, INGE HOHL,
and DAVID
G. ROBINSON
Pflanzenphysiologisches Institut der Universitat Gottingen, Untere Karspiile 2, D-37073 Gottingen, Germany Received June 15, 1994 . Accepted September 5, 1994
Summary
Legumin, vicilin and pea albumin are the most prominent storage proteins in pea seeds. In order to localize these proteins in the developing cotyledons we have employed an improved fixation procedure for immunogold labelling for electron microscopy. Profiles of protein bodies (PB, 1-2 Ilm diameter) label heavily and specifically with antibodies against vicilin and legumin. By contrast, such PB are only weakly labelled with antibodies against pea albumin 1. Antibodies against pea albumin 1 label a narrow, granulated zone at the luminal borders of the deposits. Depending on their size, storage protein deposits at the tonoplast of the protein storage vacuole (PSV) show different labelling patterns: small deposits label positively only with vicilin antibodies; labelling with legumin is seen in large deposits but is restricted to the tonoplast rather than luminal borders of the deposits. We frequently observed profiles of storage protein deposits at the tonoplast of the PSV, which were elongated and protruded into the cytoplasm. A gradient of legumin labelling in these deposits was seen. We interpret such images as stages in the formation of PB, which apparently arise by budding at the surface of the PSV.
Key words: Legumin, pea albumin, protein body, protein deposition, stratification, vicilin. Abbreviations: BSA-C = acetylated BSA; PA 1 = pea albumin 1; PB = protein body(ies); PSV = protein storage vacuole(s); TBS = tris buffered saline. Introduction
Plants, unlike animals, can store proteins as part of a strategy to provide the plant with sufficient reserves of amino acids during seed germination or at the begin of the growing season in the case of perennial woody plants. These proteins are usually found in membrane-bound vesicles termed protein bodies (PB), whose size, morphology and origin in the cell is not uniform (Pernollet, 1978; Lott, 1980). In many cereals the PB arises by direct vesiculation of the rough endoplasmic reticulum, e.g. in maize (Larkins and Hurkman, 1978) and rice (Bechtel and Juliano, 1980) endosperms. By contrast, in the cotyledons of legumes PB are formed from the protein storage vacuole (PSV), which according to some authors is a transformed vegetative vacuole, and to others has developed de novo out of the endoplasmic reticulum (see Robinson et al., 1995 for a discussion of these two possibilities ). © 1995 by Gustav Fischer Verlag, Stuttgart
The proteins stored in PB differ considerably in their structure and properties. As a general rule it is the water and salt insoluble but alcohol or acid soluble prolamins and glutelins that constitute the PB of cereals (Shewry and Tatham, 1990), and the water soluble albumins and salt soluble globulins (Shotwell and Larkins, 1988) that are characteristic of the legume PB. Normally the content of cereal and legume PB is electron opaque and stains homogenously, with only few exceptions, e.g. the maize PB (Lending and Larkins, 1989). However, the synthesis of storage proteins is known to be developmentally regulated with some polypeptides being synthesized and deposited in the PB before others (Higgins, 1984). This is particularly well exemplified in the maturing pea cotyledon, where it has been established that the synthesis of vicilin proceeds that of pea lectin, convicilin and legumin (Chrispeels et al., 1982; Gatehouse et al., 1982; Higgins et al., 1983; Millerd et al., 1978; Spencer et al., 1980; Wenzel et al., 1993). One might therefore ask whether or
438
GISELBERT HINZ, BIRGIT HOH, INGE HOHL,
and DAVID G.
ROBINSON
a
PS¥
c Fig. 1: Immunogold detection of vicilin (a), legumin (b), and pea albumin (c, d) in storage protein deposits held within protein storage vacuoles (PSV) or protein bodies (PB). Arrowheads in (c) indicate a peripheral distribution of granular substances that colocalize with the PA-l antigen. (a) x 43.500; (b) x 35.500; (c) x 28.000; (d) x 35.000; bars throughout = 0.5 Jlm.
Protein stratification
439
.,
~
... _ - - - -
a
.
~I
;""1
i
~.
:;
Fig. 2: Immunogold detection of vicilin (a) and legumin (b) in young PSV. Arrows point to clumps of storage protein at the periphery of the PSV. The larger arrows in (b) indicates that, in contrast to the other clumps, the deposition of legumin in this clump has started. In (a) the PSV is surrounded by a vicilin-positive cisternal compartment similar to those previously described by Craig and Goodchild (1984). (a) x 38.750; (b) x 39.200; bars = 0.5 J!m.
440
GISELBERT HINZ, BIRGIT HOH, INGE HOHL, and DAVID G. ROBINSON
not the various storage polypeptides are stratified into concentric zones in the pea cotyledon PB. Using immunogold labelling we provide here an answer to this question. Material and Methods
Plant material As previously given (Hoh et al., 1995; Robinson et aI., 1995).
Immunocytochemistry One mm thick slices of cotyledon tissue were left to stand for 12-16h at 4°C in primary fixative (2 % w:v paraformaldehyde + 1 % v: v glutaraldehyde in 50 mM cacodylate buffer pH 7.4). After washing 3 x 10 min in cold cacodylate buffer the tissue was immersed in secondary fixative (1 % w:v OS04 + O.S% w:v potassium hexaferrocyanate in 50 mM cacodylate buffer, pH 7.4) for 2 h at room temperature. The tissue was then washed 2 x 10 min in cacodylate buffer and 2 x 10 min in H 20 before removing excess aldehyde through a borohydride (0.5% w:v KBH4; 30 min, RT) treatment. After dehydrating in ethanol the tissue was embedded in London Resin White (hard grade), using heat polymerisation (60°C; 20 - 24 h). Thin sections were picked up on formvar-coated nickel grids and then floated on blocking solution (3 % BSA + 0.2 % BSA-C in TBS) for 30 min before exposing to the primary antibody solution for 1 h at room temperature. After washing (4 x 5 min in 1 % BSA + 0.07 % BSA-C in TBS) the sections were incubated for 1 h in 10nm gold conjugated secondary antibody solution (diluted 1: 30 in 1 % BSA, 0.07 % BSA-C in TBS). The grids were washed again (3 x 5 min in 1 % BSA, 0.07 % BSA-C in TBS; 2 x 5 min in H 20; 10 min in 1 % glutaraldehyde; 3 x 5 min in H 20) before poststaining in uranyl acetate and lead citrate. Sections were observed in a Philips CM 10 electron microscope operating at sokV.
Primary antibodies and their dilution Polyclonal antibodies against three different storage polypeptides from pea cotyledons were employed: - affinity purified antibodies raised in rabbits against legumin (Casey, 1979), diluted 1: 100-200 in 1 % BSA + 0.07% BSA-C in TBS. - affinity purified antibodies raised in rabbits against vicilin (Croy et aI., 19S0), diluted 1:4 in 1 vol 1 % BSA + 0.07% BSA-C in TBS; 2 vol fresh pea leaf extract (Melroy and Herman, 1991). - affinity purified antibodies raised in goats against the sulfur-containing albumin PA 1 (Higgins et aI., 1986), diluted 1: 4 in 1 vol low fat milk; 2 vol fresh pea leaf extract.
Reagents for immunocytochemistry Acetylated BSA (BSA-C) was purchased from Aurion, Biotrend (Cologne, Germany). Gold coupled secondary antibody conjugates (GARG, RAGG) were obtained from Biocell (Cardiff, UK). Grids and LR-White were from Plano (Marburg, Germany). Results
Immunolocalization of vicilin and legumin In thin sections of pea cotyledon cells it is easy to recognize storage protein deposits because of their electron dense character. A positive and very dense labelling with gold coupled vicilin antibodies was found for PB and for PSV in all stages of development (Figs. 1 a, 2 a). By contrast, whereas PB were also heavily labelled by legumin antibody-gold conjugates (Fig. 1 b) small deposits of storage proteins at the periphery of young PSV were free of gold particles (Fig. 2 b). Larger clumps of storage proteins in older PSV did, however, show
Fig. 3: Immunogold localization of legumin in large storage protein deposits at the periphery of PSV. Gradients of legumin incorporation are indicated by arrows. (a) Median section through a protruding clump of storage proteins; x 34.500. (b) Tangential sections through protein clumps indicating the blebbing of PB, x 40.600. Bars = 0.5/lm.
Protein stratification
441
CYTOSOL
TP~_ PSV
Fig.4: Schematic representation of the events leading to the production of a protein body (PB) in pea cotyledons. The temporal difference in vicilin (first) and legumin (second) depositions is indicated accordingly.
!
VICILIN
a positive labelling for legumin. This was restricted to that part of the deposit adjacent to the tonoplast rather than the regions facing the lumen of the PSV (Figs. 1 b, 2 b). In very large storage protein clumps, which protruded into the cytoplasm, there was a clear gradient in immunogold labelling with legumin antibodies (Fig. 3 a). Profiles of such clumps were frequently obtained, which gave the impression that PB arise by budding of storage protein clumps from the periphery of the PSV (Fig. 3 b).
Immunolocalization ofpea albumin 1 (PA 1) In a previous report Craig (1986) described that PA 1 is restricted to the outermost layers of storage protein deposits, which are found only in cells of the embryonic axis of pea embryos. We confirm here this peculiar labelling pattern (Fig. 1 c), but have seen that it also occurs in some, but not all, of the cotyledon cells. It also only seems to be present when the periphery of such protein deposits has a granular substructure (Fig. 1 c). When this is not visible anti-PA 1 immunogold labelling is randomly distributed in the storage protein depositis at the surface of the PSV, albeit at lower densities than with anti-vicilin or anti-legumin. This is to be expected since PA 1 is a much less abundant storage protein. PB at this stage show only very little labelling (Fig. 1 d). This we interpret as reflecting the fact that albumin is synthesized late in pea cotyledon maturation and PB that have already budded off will therefore not contain albumin.
Discussion
The major storage proteins in peas are the 11 S legumin and 7S vicilin globulins (Derbyshire et ai., 1976). In addition there are smaller amounts of albumins (Croy et al., 1984), and lectin (Higgins et ai., 1983). With the exception of some albumins, which have no signal peptide and remain in the cytosol (Harris and Croy, 1986; Higgins et ai., 1987), all of these polypeptides are synthesized at the rough endoplasmic reticulum where they enter the en do membrane system co-
~
LEGUMIN
translationally (Miintz, 1989), and subsequently accumulate in PB (Pernollet, 1978; Lott, 1980). The kinetics of mRNA transcription and translation in relation to storage protein formation have been followed on numerous occasions by several groups (Higgins, 1984; Wenzel et ai., 1993). Agreement exists as to the following temporal order for storage protein synthesis: vicilin - lectin - convicilin - legumin - albumin. Since there is no evidence for more than one type of PB in maturing pea cotyledons one might expect the individual polypeptide types to accumulate in the PB in sequential, zonal manner. Our results indicate that this is to a certain extent true for the major storage proteins vicilin and legumin. A parallel situation is encountered in the mature PB of maize endosperm where the lightly staining central core is comprised of a-zeins and the darkly staining outer shell has principally (3- and 'Y-zeins (Lending and Larkins, 1989). A comparison of young (lying in outermost endosperm layers) and old (lying in more centrally located cells) PB in maize suggested the a-zeins penetrate the (3- and 'Y-zeins, which are synthesized earlier, and in young PB make up the uniform darkly staining content. However, in contrast to the maize zeins, vicilin and legumin intermix, the mature PB in pea cotyledons giving no indication of a sequential accumulation of storage proteins. Our results also indicate that at least some of the PB arise from the PSV by budding or blebbing (schematically presented in Fig. 4). The contrasts with the idea that the PSV gradually accumulates storage proteins and then fragments or subdivides into large numbers of PB (see Robinson et ai., 1995, for a discussion). The latter concept has been made especially attractive because it provides an explanation to the problem of dealing with excess membrane in the PSV delivered by vesicle-mediated storage protein transport (Craig et ai., 1979). Our model for PB formation is, in this respect, equally compatible with the need to accomodate a flow of membrane to the PSV. However, as we have shown the (macro) vesiculation event leading to the formation of a PB in pea cotyledons is associated by a differential deposition of storage polypeptides. Two questions arise as a consequence of this alternative hypothesis. Firstly, why do storage proteins aggregate in clumps more or less regularly spaced along
442
GISELBERT HINZ, BIRGIT HOH, INGE HOHL, and DAVID G. ROBINSON
the tonoplast of the PSV? Secondly, do these proteins accumulate at these loci from within the PSV or are they the result of a specific fusion of storage protein-containing vesicles at predetermined sites? Acknowledgements
We gratefully acknowledge the receipt of antisera from Drs. R. Casey (Norwich, UK), R. R. D. Croy (Durham, UK) and T. J. V. Higgins (Canberra, Australia). We thank Sibille Hourticolon for photographic work and Heike Freundt for help in typing the manuscript. Bernd Rauffeisen prepared Fig. 4. This work was supported by funds from the Deutsche Forschungsgemeinschaft.
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