P l a n t a 9 Springer-Verlag 1986
Monoclonal antibodies to plant plasma-membrane antigens P.M. Norman, V.P.M. Wingate, M.S. Fitter and C.J. Lamb* Plant Biology Laboratory, Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92138, USA
Murine monoclonal antibodies to membrane antigens were generated by immunization with a crude cellular membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. From a panel of thirteen monoclonal antibodies, seven were found to be directed against antigens present on the plasma-membrane by immunofluorescence visualization of antibody binding to the surface of isolated protoplasts. The corresponding set of plasma-membrane antigen(s) were present in root, shoot and leaf tissue and some but not all of these antigens were of wide species distribution, being found in Nicotiana tabacum L., N. plumbaginifolia L., Glycine max L., Phaseolus vulgaris L. and Triticum aesfivum L. Topologically specific labeling of intact protoplasts with a monoclonal antibody reactive with an epitope present on the plasma-membrane specifically labeled a membrane fraction which equilibrated at a density of 1.14 kg/1 following centrifugation in a sucrose gradient. In addition to use as biochemical markers for fractionation and molecular characterization of plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations. Abstract.
Key words" Cell surface - Hybridoma - Immunofluorescence microscopy - Monoclonal antibodies - Plasma-membrane - Protoplast (plasma-membrane).
of important cellular functions including cellulose synthesis and wall deposition (see review by Northcote 1977), hormone transport and action (see reviews by Goldsmith 1977; Rubery 1981), transport of ions and metabolites (Spanswick 1981), as well as recognition and response to symbiotic and pathogenic microorganisms (for reviews, see C l a r k e and Gleeson 1981; Keen 1982). However, investigation of the molecular properties of plant plasmamembranes has been severely hampered by the lack of biochemical markers and attendant difficulties in membrane fractionation (for reviews, see Hall 1983; Hall and Taylor 1981; Quail 1979). Monoclonal antibody technology allows the generation of specific antibodies starting from impure, heterogeneous immunogen preparations (K6hter and Milstein 1975) and has proved to be a valuable and powerful tool in the study of the structure, function and biogenesis of the plasmamembrane of animal cells (for reviews, see Hosking and Georgiou 1982; Williams 1980). Monoclonal antibodies to epitopes present in plant endomembrane proteins (Bolwell and Northcote 1983) and epitopes putatively associated with a membraneous binding site for naphthylphthalmic acid, an inhibitor of auxin polar transport (Jacobs and Gilbert 1983), have recently been described. We describe here the generation, identification and characterisation of monoclonal antibodies to epitopes present on the plasma-membrane of plant cells. In addition to use as biochemical markers for fractionation and molecular characterisation of plant plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations.
The plasma-membrane (cell-surface membrane, plasmalemma) of plant cells has a key role in cell division, differentiation and development (for a review, see Lamb 1981), and is the site of a number
M a t e r i a l s and m e t h o d s
* To whom correspondence should be addressed
by subculture every two weeks into fresh UM1 medium (Uchi-
Plant tissue cultures. Liquid cell-suspension cultures of Nicotiana glutinosa L., initiated from stem explants, were maintained
P.M. Norman et al. : Monoclonal antibodies to plant plasma-membrane antigens miya and Murashige 1976). The sources and maintenance of other plant cultures were as previously described for Glycine max L. (Gamborg 1975); Phaseolus vulgaris L. (Dixon and Fuller 1978); Petunia hybrida L. (Uchimiya and Murashige 1976); Nieotiana plumbaginifolia L. and Nicotiana tabaeum L. : MS medium (Murashige and Skoog ]962) supplemented with 0.5 rag/1 2,4-dichlorophenoxyacetic acid and 0.5 mg/1 6-benzylaminopurine; Tritieum aestivum L. : MS medium supplemented with 146 rag/1 glutamine, 1 rag/1 naphthalene-l-acetic acid and I rag/1 2-isopentenyladenine. Tobacco plants were grown under a 16-h photoperiod in vitro in Magenta jars containing 100 ml MS medium containing 3% sucrose and 0.8% agar, following explantation of sterile nodal sections.
Membrane preparation. Cells were extracted at 4 ~ C in 25 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HCl pH 7.4 containing 0.25 M sucrose, 3 m M ethylenediaminetetraacetic acid (EDTA) and 5 mM dithiothreitol (4 ml/g FW of cells), using a pestle and mortar. The extract was clarified by filtration through Miracloth (Calbiochem, La Jolla, Ca., USA) and debris were removed by centrifugation at 1500 gav for 10 rain. The supernatant was centrifuged at 100000 g,v for I h and the pellet resuspended in phosphate-buffered saline (PBS) to give the total cellular membrane preparation. Protein content was measured by the method of Lowry et al. (1951). Monoclonal antibody production. Balb/c mice (12 weeks old, bred by the Salk Institute Animal Facility) were immunized by injection with the total cellular membrane preparation. Immunization consisted of three intraperitoneal injections of 2 mg protein in 0.5 ml of PBS, at weekly intervals, followed a week later by a single intravenous injection of I mg protein in 0.25 ml PBS. Four days after the final injection, the mice were sacrificed and splenic lymphocytes fused with $I 94/5.XXO.BU1 myeloma cells as described (Trowbridge 1978). Following fusion, the cell suspensions were plated into 24-well Linbro plates (Flow Labs, McLean, Va., USA) and grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% horse serum, under 100 laM hypoxanthine, 10 laM thymidine, 2 laM deoxycytidiue, 1 gM aminopterin (HAT) selective conditions (Litttefield 1964). Actively growing hybrid colonies were expanded in D M E M 10% horse serum supplemented with hypoxanthine, thymidine and deoxcytidine at the above concentrations (HT medium) to 2.106 cells and frozen in liquid nitrogen. Colonies secreting antibodies against the immunogen, as determined by radioimmunoassay, were cloned in HT medium by limiting dilution at 2 and 10 cells per well in the absence of feeder cells. Secreting clonal cell lines were expanded and cryopreserved, their culture medium collected for further analysis, and selected lines used to produce ascites fluid in syngeneic mice.
Radioimmunoassay of antibody binding. All steps were performed at 4 ~ C. Total cellular membrane preparations were diluted to 0.5 lag membrane protein/ml in PBS containing 0.1% sodium azide and 1 mg/ml bovine serum albumin (BSA) (PBSA-BSA) and incubated with appropriate hybridoma culture supernatants for 45 rain in 96-well V-bottom polystyrene plates (Dynatech Laboratories, Alexandria, Va., USA) (50 lal of membrane preparation and 50 lal of culture supernatant per well). The plates were then washed three times by centrifugation at 1 100 gav for t0 min, aspiration of the supernatant, resuspension of the membrane pellet using an orbital shaker, and addition of 150 lal PBS-A-BSA per well. Membranes were then incubated for 45 min with 125I-labeled rabbit anti-mouse antibody (prepared by the chloramine T method (Greenwood et al. 1963) with carrier-free lZ~I from Amersham, Arlington Heights, I1., USA) diluted in PBS-A-BSA (50 lal containing 500000 cpm per
well). The plates were washed three times as above, and the final membrane pellet was then washed into transfer tubes for ?-counting. Control assays in which the first antibody was replaced with either D M E M or monoclonal antibodies to bacterial surface antigens typically gave background values of 1000-1 500 cpm.
lmmunoJTuorescence visualisation of antibody binding to protoplast and cell surfaces. Protoplasts were prepared from suspension-cultured ceils of N. glutinosa by incubation for 2.5 h in 1% cellulase Onozuka RS and 0.1% pectolyase Y23 (Kanamatsu Gosho, Los Angeles, Ca., USA) as described by Nagata et al. (1981), with 0.4 M sorbitol and 3.3 mM 2-[N-morpholino]ethanesulfonie acid (Mes), pH 5.5, as osmoticum. Protoplasts were filtered through cotton wool to remove debris and washed by centrifugation at 100 g for 2 rain followed by resuspension in 0.4 M sorbitol buffered to pH 7.2 with 0.2% N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes). All subsequent steps were performed at 4 ~ C. Isolated protoplasts were incubated for 10 min with goat 7-globulins (1 mg/ml) to reduce non-specific binding of antibodies. Pre-treated protoplasts were then incubated for 45 min with hybridoma supernatant, the osmotic potential of which had previously been adjusted by addition of one part five fold concentrated osmoticum to four parts hybridoma supernatant. The protoplasts were washed two times by centrifugation at 100g for 2 min and resuspended in 0.4 M sorbitol, 0.2% Hepes, 1 mg/ml BSA and 0.1 mg/ml goat ?-globulins, pH 7.2 (immunofluorescence wash), and then incubated for 45 min in fluorescein-labeled goat anti-mouse antibody (Cooper Biomedical, Malvern, Pa., USA) diluted 1:10 in immunofluorescence wash. Protoplasts were washed three times prior to examination of aliquots of the protoplast suspension using a Nikon Diaphot epifluorescence inverted microscope. The excitation wavelength was 460485 nm; fluorescence was observed through a barrier filter opaque to wavelengths less than 515 nm. Mesophyll protoplasts from leaves ofN. tabacure were isolated as described by Haberlach et al. (1985), except that the osmoticum was 0.4 M sucrose, and then treated as above. For immunofluorescence visualization of binding it is essential that protoplasts are in good condition: residual cell-wall material or clinging debris bind antibodies non-specifically in the presence of sorbitol. Fixation of protoplasts with 4% formaldehyde or 0.3% glutaraldehyde in 0.4 M sorbitol resulted in non-specific antibody binding and autofluorescence, respectively. However, little non-specific binding of antibody was observed when protoplasts were fixed with 4% formaldehyde in 2.5% KCI, 1% MgSO4"7 H20, 10 mM Hepes, pH 7.2 (saline osmoticum). For study of antibody binding to intact cells of N. glutinosa the same procedure was followed save that enzymic digestion was omitted and saline osmoticum was used since sorbitol caused non-specific binding to the cell wall even with unfixed cells.
Antigen distribution in density gradients. Total cellular membranes prepared as above from 50 g FW of suspension-cultured N. glutinosa cells were centrifuged for 18 h at 100000 g~v in a 1 5 4 5 % (w/w) sucrose gradient containing 2.5 mM Tris-Mes, 0.5 mM E D T A and 1 mM dithiothreitol at pH 7.4. Gradient fractions were diluted 10-fold in PBS and aliquots absorbed to nitrocellulose filters using a slot-blot manifold (Sehleicher and Schuell, Keene, N.H., USA). Each slot was blocked by treatment with PBS containing 5% BSA for 30 rain and then incubated for I h with 40 lal hybridoma supernatant. Filters were washed three times by incubation for 30 rain in PBS containing 0.05% polyoxyethylene sorbitan monolaurate (Tween 20) and the remaining protein binding sites blocked by incuba-
P.M. N o r m a n et al. : Monoclonal antibodies to plant plasma-membrane antigens
Table 1. Characterisation of monoclonal antibodies to cellular membrane antigens from suspension-cultured cells of Nicotiana glutinosa. Antibody binding to total cellular membranes from suspension-cultured N. glutinosa cells was measured by radioimmunoassay (RIA) and data are expressed as a ratio of the bound radioactivity with test antibody from supernatants to the corresponding clonal hybridoma cell line grown in large volume, divided by the bound reactivity with control, unreactive antibody VW40.2B2 (RIA: Column 2). Antibody binding to the surfaces of protoplasts (Column 3) and cells (Column 4) from suspension cultures of N. glutinosa was monitored by immunofluorescence microscopy and is recorded on a scale of - (no binding), + / - , +, + + , + + + (strong binding). R1A of antibody binding to total cellular membranes derived from N. glutinosa protoplasts (Column 5) and from a variety of cell cultures (Columns 6~11) is recorded on the same scale, n.t. = not tested Clone
Antigen distribution (RIA)
16.1B3 16.2C6 16.3C1 16.4A6 16.4B4 17.3A3 17.3A6 17.3B4 17.3D4 17.4B5 17.4C5 23.1D5 23.2D3
4.17 3.73 3.68 3.35 4.56 2.50 2.17 3.52 2.99 3.59 4.38 5.21 3.37
+++ +++ +++ +++ + + + +/-+/+ + + + + +/+/-
+/-+ + + --+++
+++ +++ +++ +++ + + + n.t. n.t. + + + +/+ + + +++ +
+++ +++ +++ +++ + + + + + + + + + + + + + + + + + + + +++ +++
+++ +++ +++ +++ + + + n.t. n.t. + + + + + + + + + + + + +++ ++
+++ +++ +++ +++ + + + n.t. n.t. + + + + + + + + + + + +++ +++
++ ++ +++ ++ + n.t. n.t. + + + +/+/+/+++ +++
+ ++ ++ +++ + n.t. n.t. + + +/+/+/+++ +++
++ ++ ++ ++ + + + + + +/+/+/+ +/+ ++
tion in PBS containing 5% BSA prior to incubation for 2 h with ~2SI-labeled rabbit anti-mouse antibody in PBS containing 5% BSA. Filters were washed five times by incubation in PBS containing 0.05% Tween 20 for 1 h, dried, and exposed to Kodak XRP-1 film (Eastman-Kodak, Rochester, N.Y., USA) for 12 h using D u p o n t (Wilmington, Del., USA) Cronex intensifying screens. Antibody binding was quantified by scanning densitometry of the resultant autoradiograms. For topologically specific labeling, intact protoplasts were prepared, treated with monoclonal antibody and washed as described above for immunofluorescence studies, prior to treatment with 125I-labeled rabbit anti-mouse antibody (5-107 cpm per 107 protoplasts). Protoplasts were broken using a Tenbroek homogeniser (Bellco Glass, Vineland, N.J., USA). As a control, protoplasts were broken prior to incubation with the monoclonal antibody, and the total cellular membranes collected by centrifugation at 100000 g~v for 1 h before treatment with 12sIlabeled seond antibody. Following labeling, total cellular membranes were collected by centrifugation at 100000 g,v for I h and fractionated by sucrose-density-gradient centrifugation as described above. Radioactivity in gradient fractions was determined by 7-counting.
Production of monoclonal antibodies. Balb/c mice were immunized with the total cellular membrane preparation from suspension-cultured cells of N. glutinosa, following a schedule similar to that previously used in the generation of monoclonal antibodies to lymphocyte plasma-membrane antigens.
In the absence of a suitable biochemical marker for the plasma-membrane of plant cells, a crude total cellular membrane preparation was used as immunogen so as to ensure that the antigens of interest were not lost during a prior fractionation process. Furthermore, by the use of undifferentiated suspension-cultured cells as a source of immunogen it was hoped that at least some of the antibodies generated would be directed against antigens widely distributed with respect to both cell type and plant species. Only hybridoma cultures giving supernatants that exhibited a test-to-control ratio of greater than three in the radioimmunoassay were chosen for cloning. Following cloning by limiting dilution, thirteen cell lines were obtained that stably secreted antibody (Table 1).
Identification of monoclonal antibodies to plasmamembrane epitopes. In order to determine which of these antibodies were directed against antigens present on the plasma-membrane, a second screen was employed based on antibody binding to the surface of protoplasts isolated from N. glutinosa suspension-cultured cells. This procedure requires elimination of background fluorescence associated with either autofluorescence of the protoplast preparation or non-specific binding of antibody.
P.M. Norman et ai. : MonoclonaI antibodies to plant plasma-membrane antigens
Fig. 1. 1-Sa, b. Indirect immunofluorescence visualization of the binding of monoclonal antibodies to plant protoplast and cell surfaces. Micrographs were taken under (a) bright-field illumination and (b) epifluorescence illumination. 1-5 Protoplasts derived from suspension-cultured cells of N. glutinosa; 6--7 suspension-cultured cells of N. glutinosa; 8-9 protoplasts derived from leaves of N. tabacurn. Monoclonal antibodies were (1) VW40.2B2, (2) 17.4C5, (3) 17.4B5, (4) 16.4B4, (5) 23.2D3, (6) 16.4B4, (7) 23.2D3, (8) 16.4B4, (9) 23.2D3. VW40.2B2 is directed against a surface antigen of the phytopathogenic bacterium Pseudomonas syringae f. sp. glycinae, and by radioimmunoassay exhibits no binding to the total celIular membrane preparation from suspension-cultured N. glutinosa cells
Conditions were established in which zero background fluorescence was observed when first antibody was either omitted or was a monoclonal antibody directed against antigens present on bacterial cell surfaces (VW40.2B2) (Fig. 1). Six of the thirteen monoclonal antibodies directed against antigens present in the total cellular membrane preparation from N. glutinosa cells (17.3A3, 17.3A6, 17.3B4, 17.4C5, 23.1D5, 23.2D3) also gave either zero or insignificant levels of fluorescence in this assay (Fig. 1, Table 1). In contrast, the remaining seven monoclonal antibodies (16.1B3, 16.2C6, 16.3C1, 16.4A6, 16.4B4, 17.3D4, 17.4B5)exhibited strong binding to the protoplast surface with particularly intense fluorescence in a continuous ring at the periphery of the protoplast (Fig. 1, Table 1).
None of the seven monoclonal antibodies that bind to antigens present on the protoplast surface membrane (plasma-membrane) gave a positive signal in a parallel test using intact cells which retain the surrounding cell wall (Fig. 1). However, of the six antibodies that did not bind to the protoplast surface, two (17.3B4, 23.2D3) were found to bind to the surfaces of intact cells. It is concluded that these antibodies are directed against cell-wall material present as contaminants in the total cellular membrane preparation used both for immunization and the initial radioimmunoassay screening. The other four monoclonal antibodies (17.3A3, 17.3A6, 17.4C5, 23.1D5) bound neither to the surfaces of protoplasts nor to the surfaces of intact cells, indicating that these antibodies are directed
P.M. Norman et al. : Monoclonal antibodies to plant plasma-membrane antigens
I.> IO < w h-
- " ~t~.,,
Fig. 2. Distribution of membrane antigens following sucrose-density-gradient centrifugation of a total cellular membrane preparation from 1.2o % suspension-cultured cells of N. glutinosa. Antigen distribution in the density gradient ( o - - o ) was 1.15 monitored by reactivity with monoclonal antibodies 1.10 ~ 16.2C6 ( u - - i ) , 16.4A6 ( ~ v ) , 16.4B4 ( o - - o ) , 17.3A6 ( o - - o ) , 17.4B5 (A--A) and 17.4C5 (A A). Open symbols denote antibodies to 1.05 internal cellular membrane antigens, closed symbols < denote antibodies to antigens present on the 1.00 plasma-membrane m
a 0 n'7 Z < I 2
against antigens present on intracellular membranes. Radioimmunoassay of antibody binding to membrane preparations confirmed that two of the antibodies were directed against cell-wall material since these antibodies (17.3B4, 23.2D3) did not give a signal with membrane preparations isolated from protoplasts derived from suspension-cultured N. glutinosa cells. In contrast and as expected, substantial binding to protoplast membrane preparations was obtained with monoclonal antibodies directed against antigens present on the plasmamembrane and monoclonal antibodies directed against putative intracellular membrane antigens (Table 1). Species and organ distribution of antibody reactivity. The species distribution of these antigens was checked by radioimmunoassay of antibody binding to total cellular membrane preparations isolated from cultured cells. Some of the antigens present on the plasma-membrane of suspensioncultured N. glutinosa cells appear to be of relatively wide phylogenetic distribution being found not only in other solanaceous species and the legumes Glycine max and Phaseolus vulgaris but also in the monocotyledon Triticum aestivum, whereas other plasma-membrane antigens were not found in the cell membrane preparations from G. max, P. vulgaris or T. aestivum (Table 1). Within a mature plant, reactivity of the monoclonal antibodies to plasma-membrane antigens was observed with cell membranes isolated from all organs tested: root, shoot, young and mature leaves (data not shown). In the case of leaf tissue, the cellular localization of these antigens was confirmed by immunofluorescence visualization of antibody binding to the
surface of isolated mesophyll protoplasts (Fig. 1). With leaf mesophyll protoplasts there is an internal block of red fluorescence emitted by chlorophyll and surface binding of fluorescein-labeled antibody was manifest as a ring of yellow fluorescence resulting from admixture of the green and red fluorescence. Membrane fractionation. Following sucrose-density-gradient centrifugation of a crude cellular membrane preparation from N. glutinosa cells, the distribution of reactivity with four independent monoclonal antibodies (16.2C6, 16.4A6, 16.4B4 and 17.4B5) directed against epitopes present on the plasma-membrane exhibited the same characteristic pattern with two peaks at 1.10 kg/1 and 1.14 kg/1 (Fig. 2). This pattern of reactivity was distinctly different from that observed with two monoclonal antibodies (17.3A6 and 17.4C5) directed against antigens present only on internal cellular membranes. The bimodal distribution of reactivity with epitopes known to be present on the plasma-membrane indicates that these epitopes may also be present on an internal cellular membrane. Recently, it has been shown by immunoelectronmicroscopy that monoclonal antibodies to epitopes present on the peribacteroid membrane from Rhizobium-induced root nodules of pea also labeled both the plasma-membrane and Golgi body of uninfected plant cells (Brewin et al. 1985). Plasma-membrane and reactive internal cellular membranes were distinguished by analysis of the distribution of radioactivity following surface labeling of intact protoplasts with monoclonal antibody and 125I-labeled rabbit anti-mouse antibody prior to membrane extraction and fractionation (Fig. 3). Topological specificity of this pre-labeling
P.M. Norman et al. : Monoclonal antibodies to plant plasma-membrane antigens 100 7A
-~ 1.06 ~,
i .% rr
=o g: I
Fig. 3. Topologically specific labeling of the plasma-membrane of protoplasts derived from N. glutinosa suspension-cultured cells. Sequential treatment with monoclonal antibody and lzsIlabeled rabbit anti-mouse antibody was used to label either the accessible surface membranes of intact protoplasts prior to membrane extraction or total cellular membranes collected after protoplast breakage. The pattern of antibody reactivity was monitored by measurement of radioactivity in fractions obtained following centrifugation of equivalent amounts of the respective membrane preparations to equilibrium in sucrose density gradients: labeling of intact protoplasts ( e - - e ) ; labeling of extracted total cellular membranes ( o - - o ) ; ratio of antibody reactivity with intact protoplasts/extracted total cellular membranes (zx--zx) ; buoyant density ( )
procedure was demonstrated by the almost complete loss of reactivity using monoclonal antibody 17.4C5, thereby confirming that this antibody is directed against epitopes present exclusively on internal cellular membranes. In contrast, whilst labeling of intact protoplasts with monoclonal antibody 16.4B4 resulted in loss of reactivity with membranes banding at buoyant densities less than 1.13 kg/1, reactivity with membranes banding at 1.14 kg/1 was fully retained.
In this study we have exploited hybridoma technology to generate monoclonal antibodies to plant plasma-membrane antigens starting from a crude total cellular membrane preparation as immunogen. The two-stage screening procedure used here employs a reliable, routine initial assay to monitor antibody secretion at various stages during the cloning of hybridoma cell lines, followed by a specific second assay for identification of the subgroup of antibodies of particular interest. The other monoclonal antibodies generated, to antigens present on internal cellular membranes or cell walls, provide controls to check the specificity of antibody binding to the surface of isolated protoplasts in the second stage of screening and to check the topological specificity of surface labeling. Such controls are important since non-specific binding of polyclonal antisera to plant protoplast surfaces (Larkin 1977; Raft et al. 1980) and rapid internalization of extracellular macromolecules (Power and Cocking 1970; Tanchak et al. 1984; Williamson et al. 1976) have previously been encountered. The high proportion of monoclonal antibodies directed toward plasma-membrane epitopes (7 out of 13) might reflect the existence of immunodominant epitopes on this membrane. Recently, Anderson et al. (1984) have noted that immunization of mice with style extracts from mature flowers of Nicotiana alata resulted in a preponderance of monoclonaI antibodies directed against immunodominant epitopes present in the glycosyl component of arabinogalactan glycoproteins. However, protein immunoblotting has demonstrated that the present set of monoclonal antibodies to plasmamembrane epitopes are directed against a diverse set of epitopes residing on different plasma-membrane glycoproteins. In a subsequent series of immunizations with total cellular membrane preparations derived from suspension-cultured cells of N. plumbaginifolia, of the 70 monoclonal antibodies characterized only 7 were directed to epitopes present on the plasma-membrane (unpublished). Our data shows that some plasma-membrane antigens appear to be widely distributed both with respect to plant species and organs of the intact plant. Reactivity with appropriate monoclonal antibodies can be used directly as a marker for plasma-membrane epitopes in vitro and, in combination with topologically specific labeling of intact protoplasts these antibodies provide a rigorous and general marker to monitor fractionation of plant plasma-membranes in vitro. Using this approach we have demonstrated that plasma-mere-
P.M. Norman et al. : Monoclonal antibodies to plant plasma-membrane antigens
branes derived from suspension cultured cells of N. glutinosa characteristically centrifuge to equilibrium at a buoyant density of 1.14 kg/1 in sucrose density gradients. Treatment of protoptasts derived fiom suspension-cultured G. m a x cells with diazotized [3SS]sulfanilic acid (Galbraith and Northcote 1977) and protoplasts derived from suspension-cultured Daucus carota cells with [14C]acetyl-concanavalin A (Boss and Ruesink 1979) selectively labels membranes of this buoyant density, although in these studies there was no independent evidence to verify either reactivity with surface molecules or topological specificity. By suitable choice of immunogen, including plasma-membranes fractionated with the aid of the antibody markers described here, in conjunction with appropriate variants of the two-stage screening strategy it should now be possible to obtain monoclonal antibodies to a wide range of plasmamembrane antigens specifically associated with particular functions, cell types or plant species. Hence the monoclonal antibodies and overall experimental strategy described here may greatly facilitate study of the molecular structure, functional specialization and biogenesis of plant plasma-membranes. Furthermore, specific binding of monoclonai antibodies to the protoplast surface was obtained under conditions in which the protoplasts remained potentially viable. Thus these antibodies also provide the basis for development of novel, non-invasive, non-destructive and versatile selection tools for plant cell and gene manipulations, and in a forthcoming paper the use of these monoclonal antibodies as the basis for a general method to identify heterokaryons in protoplast fusion procedures will be described. We thank Ian Trowbridge and Derek Domingo for advice on hybridoma techniques, Ian Trowbridge for the gift of rabbit anti-mouse antibody, Bob Hyman for myeloma cell line S194/ 5.XXO.BU1, Michael Hahn for preparation of leaf mesophyll protoplasts and Evelyn Wilson for preparation of the manuscript. This work was supported by grants to C.J.L. from the McKnight Foundation, the Seaver Institute, and the U.S. Department of Agriculture, Competitive Grants Program No. 83CRCR-1-1251.
References Anderson, M.A., Sandrin, M.S., Clarke, A.E. (1984) A high proportion of hybridomas raised to a plant extract secrete antibody to arabinose or galactose. Plant Physiol. 75, 1013-1016 Bolwell, G.P., Northcote, D.H. (1984) Demonstration of a common antigenic site on endomembrane proteins of Pha-
seolus vulgaris by a rat monoclonal antibody. Tentative identification of arabinan synthase and consequences for its regulation. Planta 162, 139 146 Boss, W_F., Ruesink, A.W. (1979) Isolation and characterization of concanavalin A-labeled plasmamembranes of carrot protoplasts. Plant Physiol. 64, 1005 1011 Brewin, N.J., Robertson, J.G., Wood, E.A., Wells, B., Larkins, A.P., Galfre, G., Butcher, G.W. (1985) Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium-induced root nodules of pea cross-react with plasmamembrane and Golgi bodies. EMBO J 4, 605 6ll Clarke, A.E., Gleeson, P.A. (1981) Molecular aspects of recognition and response in the pollen-stigma interaction. Rec. Adv. Phytochem. 15, 161 211 Dixon, R.A,, Fuller, K.W. (1978) Effects of growth substances on non-induced and Botrytis cinerea culture-filtrate induced phaseollin production in Phaseolus vulgaris cell cultures. Physiol. Plant Pathoh 12, 279-288 Galbraith, D.W., Northcote, D.H. (1977) The isolation of plasmamembrane from protoplasts of soybean suspension cultures. J. Cell Sci. 24, 295-310 Gamborg, O.L. (1982) Callus and cell culture. In: Plant tissue culture methods 2nd edn., pp. 1-10, Wetter, L.R., Constabel, F., eds. National Research Council of Canada, Ottawa Goldsmith, M.H.M. (1977) The polar transport of auxin. Annu. Rev. Plant Physiol. 28, 439-478 Greenwood, F.C., Hunter, W.M., Glover, J.S. (1963) The preparation of ~31I-labeled human growth hormone of high specific radioactivity. Biochem. J. 89, 114-123 Haberlach, G.T., Cohen, B.A., Reichert, N.A., Baer, M.A., TowilL L.E., Helgeson, J.P. (1985) Isolation, culture and regeneration of protoplasts from potato and several related Solanum species. Plant Sci. 39, 67 74 Hall, J.L. (1983) Plasmamembranes. In: Isolation of membranes and organelles from plant cells, pp. 55-82, Hall, J.L., Moore, A.L., eds. Academic Press, London, New York Hall, J.L., Taylor A.R.D. (1981) Isolation of the plasmamembrane from higher plant cells. In: Plant organelles, pp. 103 111, Reid, E., ed. Wiley, New York Hosking, C.S., Georgiou, G.M. (1982) Application of monoclonal antibodies to the study of human lymphocyte surface antigens. In: Monoclonal hybridoma antibodies: Techniques and applications, pp. 177-192, Hurrell, J.G.R., ed. CRC Press, Boca Raton, FI., USA Jacobs, M., Gilbert, S.F. (1983) Basal localization of the presumptive auxin transport carrier in pea stem cells. Science 220, 1297-1300 Keen, N.T. (1982) Specific recognition in gene-for-gene hostparasite interactions. Adv. Plant Pathol. 1, 35 82 K6hler, G., Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497 Lamb, C.J. (1981) Molecular approaches to the study of cell differentiation and development in higher plants: The biochemistry of xylem and phloem production. In: Development and differentiation, pp. 14~178, Buckingham, M.E., ed. CRC Press, Boca Raton, F1., USA Larkin, P.J. (1977) Plant protoplast agglutination and membrane-bound fl-lectins. J. Cell Sci. 26, 31-46 Litttefield, J.W. (1964) Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709 710 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 Murashige, T., Skoog, F. (1962) A revised medium for rapid
P.M. Norman et al. : Monoclonal antibodies to plant plasma-membrane antigens growth and bioassays with tobacco tissue cultures. Physiol. Plan~. 15, 473-497 Nagata, T., Okada, K., Takebe, I., Matsui, C. (1981) Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Mol. Gen. Genet. i84, 161 165 Northcote, D.H. (1977) The synthesis and assembly of plant cell walls: possible control mechanisms. In: Synthesis, assembly and turnover of cell surface components, vol. 4, pp. 717-751, Poole, G., Nicholson, G.L., eds. Elsevier, Amsterdam Power, J.B., Cocking, E.C. (1970) Isolation of leaf protoplasts: macromolecule uptake and growth substance response. J. Exp. Bot. 21, 64-70 Quail, P.H. (1979) Plant cell fractionation. Annu. Rev. Plant Physiol. 30, 425-484 Raft, J., McKenzie, I.F.C., Clarke, A.E. (1980) Antigenic determinants of Prunus avium are associated with the protoplast surface. Z. Pflanzenphysiol. 98, 225-234 Rubery, P.H. (1981) Auxin receptors. Annu. Rev. Plant Physiol. 32, 569-596
Spanswick, R.M. (1981) Electrogenic ion pumps. Annu. Rev. Plant Physiol. 32, 26%289 Tanchak, M.A., Griffing, L.R., Mersey, H.G., Fowke, L.C. (1984) Endocytosis of cationized ferritin by coated vesicles of soybean protoplasts. Planta 162, 481486 Trowbridge, I.C. (1978) Interspecies spleen-myeloma hybrid producing monoclonal antibodies against mouse lymphocyte surface glycoprotein, T200. J. Exp. Med. 148, 313-323 Uchimiya, H., Murashige, T. (1976) Influence of the nutrient medium on the recovery of dividing cells from tobacco protoplasts. Plant Physiol. 57, 424-429 Williams, A.F. (1980) Cell-surface antigens of lymphocytes: markers and molecules. Biochem. Soc. Symp. 45, 27-50 Williamson, F.A., Fowke, L.C., Constabel, F.C., Gamborg, O.L. (1976) Labeling of concanavalin A sites on the plasma membrane of soybean protoplasts. Protoplasma 89, 305-316
Received 25 September; accepted 25 November 1985