Ultrastructural studies of the binding of concanavalin A to the plasmalemma of higher plant protoplasts

Summary The binding of concanavalin A to the plasmalemma of higher plants has been studied using protoplasts of two species. The lectin aggregates both tobacco (Nicotiana tabacum L.) leaf protoplasts and protoplasts prepared from a suspension cell culture of grapevine (Vitis vinifera L.). Differences in lectin binding have been investigated using concanavalin A conjugated to ferritin or bound to colloidal gold. Tobacco protoplasts exhibit continuous and saturated labelling of the plasmalemma surface with gold-concanavalin A mixtures. Vine protoplasts under the same conditions show a discontinuous and patchy distribution of label. These results are discussed in terms of a possible binding mechanism.Abbreviations ConA concanavalin A - PBS Phospholi Buffered Saline - PEG polyethylene glycol     

Labelling of concanavalin A sites on the plasma membrane of soybean protoplasts

Summary The plasma membranes of protoplasts from cultured soybean cells bind haemocyanin after treatment with concanavalin A. On unfixed protoplasts the binding sites were unevenly distributed. In thin section and in Pt/Pd replicas, the haemocyanin molecules were seen in clusters. After 16 hours incubation, the haemocyanin appeared in tighter clusters and newly synthesized cellulose microfibrils were present. In contrast, the protoplasts prefixed before concanavalin A treatment exhibited a very even distribution of haemocyanin. These observations are discussed in relation to membrane structure and the fluid mosaic model based on analogous studies of animal cells.Supported by the National Research Council of Canada.     

Interactions of galactose-binding lectins with plant protoplasts

Summary Protoplasts isolated from cell suspension cultures of carrot (Daucus carota L.) and leaves of tobacco (Nicotiana tabacum L.) were treated with three lectins specific for galactosyl residues. After incubation with RCA I (Ricinus communis agglutinin, molecular weight 120,000) conjugated to ferritin or fluorescein, freshly isolated protoplasts displayed heavy labeling of their surfaces. Moreover, they agglutinated rapidly when exposed to low concentrations of RCA I. In parallel studies, PNA (peanut agglutinin) also bound extensively to the protoplast plasma membranes whileBandeiraea simplicifolia lectin I attached relatively weakly. When protoplasts were cultured for two days and then incubated with conjugates of RCA I and PNA, additional binding sites were revealed on the regenerating walls.The results indicate that galactosyl residues are distributed densely over the surface of plant protoplasts. They also allow inferences to be made regarding the positions and linkages of the galactose groups being recognized by the lectins. Moreover, they open up the question whether the galactosyl moieties detected in the wall derive from those labeled on the plasma membrane. To conclude, we make comparisons with binding by concanavalin A, and predict that galactose-recognizing lectins will join and in certain respects prove superior to concanavalin A as probes of the plant cell surface.     

Targeting of large liposomes with lectins increases their binding to plant protoplasts

Soybean agglutinin, peanut agglutinin, and concanavalin A were covalently bound by condensation reaction to gangliosides and ceramides incorporated within the bilayer of multilamellar and unilamellar liposomes. These modified liposomes had a much higher affinity for carrot and tobacco protoplasts except when concanavalin A was used.

In addition, soybean agglutinin and concanavalin A were attached by ligand-specific binding to liposomes containing cholesterol molecules derivatized with each lectin-specific sugar. This procedure allowed efficient crosslinking of liposomes to protoplasts. The same effect was achieved with soybean agglutinin and peanut agglutinin when derivatized cholesterol was replaced by gangliosides. The implications of these findings for the liposome-mediated nucleic acid transfer into protoplasts are discussed.

     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

Morphology ofEntomophthora muscae protoplasts grownin vitro

Summary Entomophthora muscae (C.) Fres. can be grownin vitro as protoplasts. Light and electron microscopical studies of thein vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape ofE. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.Abbreviations Con A concanavalin A - DH Drosophila cell culture medium - FITC fluorescein isothiocyanate - GLEN glucose-lactal-bumin-yeast extract-NaCl culture medium for protoplasts - HBL hyphal body-like protoplasts - MM Mitsuhashi and Maramorosch' insect cell culture medium - PATAg periodic acid-thiocarbohydrazide-silver proteinate technique - PBN phosphate buffer with NaCl - S spherical protoplasts - WGA wheat germ agglutinin     

Chemical and structural alterations at the cell surface of Candida tropicalis, induced by hydrocarbon substrate.

The surface-localized polysaccharide of alkane-grown cells of Candida tropicalis was identified as mannan containing approximately 4% covalently linked fatty acids. Glucose-grown cells lacked the mannan-fatty acid complex. The surface structure of alkane-grown cells showed a radial arrangement of the wall polymers, with protruding parts. The cell surface of glucose-grown cells was smooth, with a coherent outer limit. The mannan was localized by using concanavalin A. Masking of the mannan with concanavalin A reduced the binding affinity of the surface for alkane, indicating the involvement of the surface-localized mannan-fatty acid complex in the binding of alkanes.     

Alterations in the surface glycoproteins of chicken erythrocytes following transformation with erythroblastosis strain R virus

Summary Erythroblasts from marrows of chicks infected with RNA-virus (strain Rerythroblastosis virus) were found to possess a small but consistent increase in the number of concanavalin A binding sites per cell compared to erythroblasts derived from the marrows of phenylhydrazine-treated birds. Both types of erythroblast possessed more surface glycoproteins per cell accessible to concanavalin A (Con A) than marrow and peripheral blood erythrocytes. Employment of concanavalin A conjugated to ferritin showed marked differences in the spatial arrangement of the Con A receptors between phenylhydrazine and virus-induced erythroblasts but little difference was observed in the surface density of the Con A sites between erythrocytes and erythroblasts, a result which agrees with the amount of bound labeled Con A when this data is expressed in terms of the cell surface.The amount of labeled Con A bound to erythrocytes derived from the marrow was greater than that derived from the peripheral circulation, a result which is substantiated by the ferritin Con A studies which show an increase in the density of Con A sites on the marrow blood cells. Trypsinization increases the number of sites and the agglutininability of the marrow cells.The increase in the susceptibility of the cells to agglutinate with concanavalin A paralleled the observed increase in the number of binding sites per cell.     

Orientation of the rhodopsin sugar moiety in bovine disk membrane.

Rhodopsin from the bovine rod outer segment contains a covalently linked carbohydrate moiety (Heller, J. & Lawrence, M.A. (1973) Biochemistry 9, 864--868). We studied the location of this carbohydrate moiety on the disk membrane by using ferritin-conjugated concanavalin A and concanavalin A labelled with fluorescein isothiocyanate. Electron microscopic observation of sonicated disk membrane that was labelled with ferritin-concanavalin A revealed the electron-dense image of ferritin on the inner surface of the disk membrane and not on its outer surface. Intact disk membrane that was similarly treated with ferritin-concanavalin A showed a complete absence of ferritin molecules on its surface. In an independent series of experiments we confirmed that the sonicated disk membrane bound three to five times more fluorescein-labelled concanavalin A than the intact disk membrane did. From these experiments we conclude that the carbohydrate moiety of bovine rhodopsin is located on the inner surface of the disk membrane, in agreement with the report by Rohlich on the frog rod outer segment disk membrane (Rohlich, P. (1976) Nature 263, 789--791).     

Pinocytosis and locomotion in amoebae XIV. Demonstration of two different receptor sites on the cell surface ofAmoeba proteus

Summary Two different receptor sites, located on the cell surface ofAmoeba proteus were detected by using fluorescent analog cytochemistry (FAC) and electron microscopy (EM). Bovine serum albumin labeled with fluoresceine-isothiocyanate (FITC-BSA) and unlabeled ferritin bind, in a pH-dependent manner, as cations at the outer filaments of the mucous layer. The anionic receptor sites show a high affinity for Ca-ions which suppress the binding capacity of FITC-BSA and ferritin at low pH-values. The cation receptors obviously play an important role in the initiation of pinocytosis as demonstrated by the internalization, intracellular translocation and sequestration of the FITC-BSA. FITC- or ferritin-labeled concanavalin A (FITC-Con A, ferritin-Con A) bind predominantly in a pH-independent manner at the tips of the outer filaments and the basal zone of the mucous layer. The binding capacity of FITC-Con A is not influenced by external Ca-ions. Other lectins such asDolichos bifloris agglutinin (DBA), peanut agglutinin (PNA),Ricinus communis agglutinin I (RCA I), soybean agglutinin (SBA),Ulex europaeus agglutinin I (UEA I) and wheat germ agglutinin (WGA) are not specifically bound to the cell surface. So far, no experimental evidence has been gathered for the definitive function of a Con-A receptor in the mucos layer ofAmoeba proteus.Abbreviations BSA bovine serum albumin - Con A concanavalin A - CTC chlorotetracycline - DBA Dolichos bifloris agglutinin - DTE dithioeritritol - FITC fluorosceine-isothiocyanate - IEP iso electric point - PIPES 1-4-piperazine-diethane sulfonic acid - PNA peanut agglutinin - RCA I Ricinus communis agglutinin I - SBA soybean agglutinin - Uac uranylacetat - UEA I Ulex europaeus agglutinin I - WGA wheat germ agglutinin     

Localization of the carbohydrate residue of the S-layer glycoprotein from Clostridium thermohydrosulfuricum L111-69

The location of the covalently attached carbohydrate residue of the S-layer glycoprotein from Clostridium thermohydrosulfuricum L111-69 was determined by electron microscopical procedures after converting the hydroxyl groups of the carbohydrate chains into carboxyl groups by succinylation. The introduction of carboxyl groups was examined by labelling with polycationized ferritin (PCF; a net positively charged topographical marker for electron microscopy). Cyanogen bromide was used for activating vicinal hydroxyl groups of the carbohydrate chains which could then react with amino groups of amino carbonic acids or ferritin. The amount of covalently bound ferritin was determined by freeze-etching and UV-measurement. Both, succinylation experiments and covalent attachment of ferritin confirmed that at least a considerable portion of the carbohydrate residue must be located on the S-layer surface.     

Hyphal walls of isolated lichen fungi

The hyphal walls of three mycobionts, isolated from the lichens Xanthoria parietina, Tornabenia intricata and Sarcogyne sp. were investigated by two techniques: microautoradiography of fungal colonies exposed to radioactive carbohydrate precursors; and binding, in vivo, of fluorescein conjugated lectins to hyphal walls of such colonies.N-[³H] acetylglucosamine was readily incorporated into tips, young hyphal walls and septa of the three mycobionts and the free-living fungus Trichoderma viride, but not into Phytophthora citrophthora, indicating that chitin is a major component of the mycobionts' hyphal walls. All three mycobionts, but neither of the free-living fungi, incorporated [³H] mannose and [³H] mannitol into their hyphal walls.Fluorescein-conjugated wheat germ agglutinin was bound to the hyphal walls of the three mycobionts and T. viride, but not to the walls of P. citrophthora; the binding pattern was similar to the grain pattern obtained in autoradiographs after short N-[³H] acetylglucosamine labelling. As wheat germ agglutinin binds specifically to chitin oligomers, the lectin binding tests further confirmed that chitin is a mycobiont hyphal wall component.Binding characteristics of several fluorescein-conjugated lectins to the three mycobionts indicated that this technique can yield useful information concerning the chemical composition of hyphal wall surfaces.List of abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - TCA trichloroacetic acid - PNA peanut agglutinin - LA lotus agglutinin - Glc NAc N-acetylglucosamine - ConA concanavalin A - SBA soybean agglutinin - WBA waxbean agglutinin Part of an M.Sc. thesis submitted by A. Braun to the Department of Botany, Tel Aviv University.     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans

A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from ecch species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion.Abbreviations AC alternating current - DC direct current     

Immunoferritin labeling of respiratory nitrate reductase in membrane vesicles of Bacillus licheniformis and Klebsiella aerogenes

The indirect immunoferritin labeling method was used to localize the membrane-bound respiratory nitrate reductase in membrane vesicles and protoplasts or spheroplasts of Bacillus licheniformis and Klebsiella aerogenes, respectively. For a comparison of the labeling of the various vesicle preparations, which differed not only in size but also in the percentage of inside-out orientation, a quantification of the results was needed to circumvent the problem of non-specifically bound ferritin. From the results the sidedness of the nitrate reductase in the cytoplasmic membrane of the abovementioned bacteria was determined as being cytoplasmic in B. licheniformis and as transmembranous in K. aerogenes.Non-Standard Abbreviations PBS phosphate buffered saline - IgG immunoglobulin G     

Characterization of cell surface carbohydrates on asexual spores of the water moldSaprolegnia ferax

Summary In asexual reproduction of the water mold,Saprolegnia ferax, four distinct and sequentially produced spores are involved in dispersal, two of which are motile and two of which are nonmotile. Composition of cell surface glycoproteins may be important in dispersal strategies for each of these stages. Binding patterns of fluorescently labelled lectins were investigated to identify differences in glycoproteins of asexually produced dispersal stages. The pattern of lectin binding to zoospores was diverse. FITC-Con A bound to surfaces of zoospores and membranes of the water expulsion vacuole system, indicating the prescence of mannosyl and glucosyl residues. In zoospores incubated for more than 30 min in FITC-WGA and FITC-GS II. which bind N-acetyl glucosamine, fluorescence was sometimes localized in peripheral, intracellular patches. In shorter incubations, secondary zoospores bound these lectins along the groove region where K-bodies were located. Surfaces of cystospores typically bound FITC-WGA, but not FITC-GS II. FITC-GS II, however, bound to empty cystospore walls, probably because reactive sugars were available at the inner surface of the wall. Germ tubes emerging from cystospores bound labelled WGA and GS II, but not Con A. The same lectin binding pattern was found along discharge papilla of primary cystospores, indicating that modifications in cystospore walls associated with direct germination and zoospore discharge were similar. Thus, glycoproteins involved in early establishment of the hyphal system differ from those forming the cell surface of cystospores. Differences in the binding pattern of lectins to zoospores and cystospores highlight differences between cell surface carbohydrates of motile and nonmotile asexual stages.Abbreviations BPA lectin fromBauhinia purpurea - C₁ primary cystospore - C₂ secondary cystospore - Con A concanavalin A, lectin fromCanavalia ensiformis - DBA lectin fromDolichos biflorus - DIC Nomarski differential interference contrast optics - DS dilute salts - FITC fluorescein isothiocyanate - FUC fucose - Gal galactose - GalNAc N-acetyl galactosamine - Glc glucose - GlcNAc N-acetyl glucosamine - GS I Griffonia simplicifolia lectin I - GS II G. simplicifolia lectin II - Man mannose - MPA lectin fromMaclura pomifera - PC phase contrast optics - PNA lectin fromArachis hypogaea - SBA soybean agglutinin, lectin fromGlycine max - UEA-1 lectin fromUlex europaeus - WGA wheat germ agglutinin fromTriticum vulgare - WV water expulsion vacuole     

Measurement of the testosterone binding parameters for both testosterone-estradiol binding globulin and albumin in individual serum samples

This report describes a solid phase method for the characterization of testosterone binding to both albumin and testosterone-estradiol binding globulin (TeBG). TeBG is adsorbed from serum samples onto a solid phase matrix of concanavalin A covalently linked to 4B Sepharose. The binding of testosterone is then examined both in the presence and absence of the endogenous serum albumin. Analysis of the resulting Scatchard plots permits determination of the TeBG binding capacity, TeBG association constant and a parameter of albumin binding equivalent to the product of its affinity and capacity for binding testosterone. Results showed that the TeBG capacity was lower in men than in women (18.4 +/- 5.8 vs. 33.1 +/- 19.2 nM, p less than 0.01). The association constant was greater in men (1.59 +/- 0.35 vs. 1.19 +/- 0.32 x 10(9)M-1, 10(9)M-1, p less than 0.01). There was no difference in the albumin binding parameter (43.8 +/- 18.3 vs. 46.6 +/- 15.5, NS). These parameters can then be used to calculate the distribution of the circulating testosterone into albumin bound, TeBG bound and unbound fractions.     

Lectin binding to the porcine and human ileal receptor of intrinsic factor-cobalamin

The purified porcine recpptor for the intrinsic factor-cobalamin complex bound to concanavalin A, lentil lectin and wheat germ lectin covalently coupled to Sepharose and was eluted with the corresponding soluble sugars. In contrast, human intrinsic factor bound efficiently to concanavalin A, to some extent to lentil lectin, but only slightly to wheat germ agglutinin. The binding of IF-Cbl to the receptor was inhibited when the receptor was pre-incubated with soluble wheat germ aglutinin, with an inhibition constant estimated to be 1.9 mol/l. After transfer of the purified receptor from SDS-PAGE to Immobilon, ligand blotting of the purified receptor with iodinated lectin showed that concanavalin A and lentil lectin bound to three (75, 56 and 43 kDa) components but that wheat germ agglutinin bound only to the 75 kDa component. These results showed that the subunit of the receptor could bind to wheat germ agglutinin, resulting in an inhibition of its binding with intrinsic factor. Both binding sites of intrinsic factor and of wheat germ agglutinin could be located near to each other.     

Lectin teratogenesis. II: Demonstration of increased binding of concanavalin A to limb buds of rabbit embryos during the teratogenically sensitive period

The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160 micrograms (in 40 microliter) are injected into the exocoelom on gestational days 12-15 but does not cause malformations on days 10-11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12-15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15-20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of alpha-methyl-D-mannoside (alpha MM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appeared normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein-labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of alpha MM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin-labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin-labelled con A was preincubated with alpha MM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside