Plant Protoplast Agglutination by Lectins

Abstract

Coicanavain A, soybean (Glyce max L.) ectin, castor bea (Rkhns communis L.) lectn, and peanut (Machis hypogaea L.) lectin were able to agglutinate protoplasts prepared from a variety of plant species. The seven other lectins tried were unable to agglutinate those protoplasts tested. Protoplasts prepared from 11 species were used. The lectins examined were not able to differentiate among protoplasts of different species.Recently there has been some interest in the effects of plant lectins on plant cell systems. Phaseolus vulgaris lectin was shown to have a mitogenic effect on tomato callus cells (13) and barley and pea root tip cells (17). Soybean lectin was mitogenic for soybean callus cells (9). PHA2 and Con A both stimulate Lilium pollen germination (18). Golynskaya et al. (7) found hemagglutinins in Primula stigmata which also effected pollen tube growth. Pretreatment of Gladiolus stigmata with Con A prevented compatible pollen tubes from penetrating (10). There is also considerable evidence that the specificity of host-symbiont interactions is determined by lectin binding (1,3,4,8,15,21). Gamborg and Miller (5) suggested that lectins may be useful for protoplast aggregation which in turn would facilitate fusion. Since then Glimelius et al. (6) reported the agglutination of carrot culture cell protoplasts by Con A. They observed inhibition of the agglutination by methyl-a-D-glucoside and a great reduction of agglutination using glutaraldehyde-fixed protoplasts. Low temperature (4 C) limited agglutination and higher cell densities favored higher agglutination percentages. Burgess and Linstead (2) confirmed the ability of Con A to interact with protoplast surfaces. Using Con A conjugated with colloidal gold they demonstrated both specific binding (inhibited by 0.1 M a-methyl mannoside) to plasmalemma of Vitis vinifera culture and tobacco leaf protoplasts, and also nonspecific binding to amorphous material near the plasmalemma. The ability of plant lectins to interact with plasmalemma may be of significance to their physiological role. Ruesink (16) and Williamson et aL (19,20) (200 g, uncooked) were milled, defatted with 500 ml of petroleum ether, and air-dried. A suspension in 0.9% (w/v) NaCl was stirred for 3 hr and the insoluble matter removed twice by centrifugation. (NH4)SO4 was added to 60% (w/v) saturation and the centrifuged pellet redissolved in 110 ml of 0.9%/v (w/v) NaCl and dialyzed against 0.9%o (w/v) NaCl for 24 hr with one change of saline. One sample was also dialyzed against OSI (pH 7.2) for 24 hr.Vicia faba beans (40 g) were milled, defatted with 300 ml of petroleum ether, and dried in air. The flour was suspended and stirred for I hr in 200 ml of 0.9% (w/v) NaCl. The centrifuged sediment was discarded. The supernatant was fractionated by (NH4)SO4 precipitation into the 0 to 48%, 48 to 65%, 65 to 80%o and 80 to 100%o (w/v)

Structural Requirements for the Binding of Phenylglycosides to the Surface of Protoplasts

“…Previous studies of phenyl-pl-glycoside binding to protoplasts (1 1, 12, 17) have generally relied on Yariv antigens as probes. Indirect evidence that simple phenyl-,B-glycosides may also bind to the protoplast surface and to soluble AGPs has come from observations that pnitrophenyl-fl-D-glucoside (12,17), salicin (5,12), certain other phenyl-fl-glycosides (12), and a flavonol glycoside (9) can inhibit Yariv antigen-induced protoplast agglutination or AGP precipitation. Relatively high concentrations (10-150 mM) of these simple phenylglycosides are required, however, to achieve this inhibition.…”

“…Although the plasma membrane components responsible for this binding have been called ,B-lectins (10)(11)(12), it is now clear that these molecules (probably some form of AGP) should not be thought of as lectins in the usual sense. Although Yariv antigens interact with these membrane components to induce tight protoplast agglutination, thioYariv antigens and other mono-and poly-phenyl-f3-glycosides have no detectable affinity for the protoplast surface.…”

“…Larkin (11,12) subsequently showed that protoplasts from a wide variety of plants could be agglutinated by many of the same Yariv antigens that precipitated AGPs. Recently, Samson et al (17) have shown that a plasma membrane fraction from Phaseolus hypocotyls contains AGP, as judged by the presence of hydroxyproline and the staining of a low pI component by a Yariv antigen.…”

Plant Plasma Membrane Proteins

“…In order to obtain additional evidence that the 75 kD antiserum was directed against epitopes present on the plasma membrane, an immunofluorescence assay was used. Because animal IgG fractions were shown to bind to plant protoplast surfaces- (21,26,28) cumvented. Figure 4 demonstrates that the 75 kD antiserum was effective as a protoplast surface label.…”

The uses of isolated protoplasts in plant genetics

“…Various methods have been suggested which would minimise the latter intra-strain fusions. Melchers (1977a) describes work in progress to attempt to give the protoplasts to be fused opposite charges, but reports no success, and it is possible that the work of Larkin (1977Larkin ( , 1978a on protoplast agglutination might lead to some technique for the maximisation of interstrain fusion.…”