Biochemical differentiation in the tobacco flower probed with monoclonal antibodies

Abstract

We have isolated a series of monoclonal antibodies that react to antigens in flowers of Nicotiana tabacum L. (tobacco) displaying specificity or preferentiality in their cell and tissue distributions. We immunized mice with extracts from tobacco flowers and then screened the hybridomas by enzyme-linked immunosorbent assay (ELISA) against extracts from leaves, sepals, petals, stamens and pistils; twenty five were chosen from the total screened. The antigens detected by about half of the antibodies were periodate-sensitive, implying that the epitopes were carbohydrate. Competition ELISA assays were used to determine if any antibodies were reacting to the same epitopes. Western blot analysis showed that while some antibodies reacted to specific bands, the bulk either failed to react or reacted to multiple bands, consistent with a glyco-conjugate nature for many of the antigens. Analysis of the spatial pattern of antigen distribution within tobacco flowers by immunolocalization showed that some antibodies recognized epitopes that were limited to very specific cells and tissues. We used the immunolocalization technique to analyze a mutant with stigmoid anthers: an antibody recognizing a pistil transmitting-tract antigen also reacted to cells in stigmoid anthers. Our results with this antibody set imply that biochemical differentiation within the tobacco flower includes cell-and tissue-specific glyco-moeities, and also that similarities, at the biochemical level, exist between a normal floral organ and the abnormal organ in a phenotype with a developmental switch.

Developmentally regulated antigen associated with calcium crystals in tobacco anthers

“…The antibody NtF-8B1 reacts to an antigen in the tobacco anther that localizes in cells scattered throughout the connective tissue, and a small group of peripheral cells just beneath the epidermis (Evans et al 1988). Figure 1E shows an immunolocalization of the antibody, using an FITC-conjugated secondary antibody for detection.…”

“…Methods were used for immunolocalizations that had been previously developed by Evans et al (1988). Briefly: sections on gelatin-coated slides were treated with goat serum for 30 rain, then antibody NtF-8B1 (purified and used at 7 lag" ml-1) for 2 h. The slides were washed three times for 10 rain each in Tris (2-amino-2-hydroxymethyl-1,3-propanediol) buffered saline (20 mM Tris, 0.9 % NaC1, pH 7.2) and treated with goat serum for 30 min.…”

“…Recently, Evans et al (1988) used crude extracts of tobacco flowers to generate a series of monoclonal antibodies directed against subsets of floral cells, tissues, and organs. These antibodies can yield insights into the basic processes of cell and tissue differentiation.…”

Arginine Decarboxylase of Oats Is Clipped from a Precursor into Two Polypeptides Found in the Soluble Enzyme

“…To generate an antibody that recognized nondenatured protein, we purified arginine decarboxylase using nondenaturing preparative PAGE and then sampled the enzyme activity in each gel slice. Mice were immunized with the partially pure extracts, and hybridomas were generated by standard methods (6,9). Media from individual hybridomas were screened by an immunoprecipitation assay to test for monoclonal antibodies that would immunoprecipitate arginine decarboxylase activity; one line, As-8B5, was positive.…”

“…Western blots were performed essentially as described previously and by others (6,9). After the blots were transferred to nitrocellulose, they were blocked in 4% BSA plus 1% fish skin gelatin overnight at 40C.…”