Monoclonal antibodies against phloem P‐protein from plant tissue cultures. I. Microscopy and biochemical analysis

Most research involving phloem proteins is done with phloem exudates, which are not easily obtained from many plants. We report here on the use of tissue cultures to study phloem proteins. Monoclonal antibodies against the filamentous phloem protein, P-protein, were made by injecting mice with a phloem-enriched fraction isolated from Streptanthus tortuosus callus grown on a medium that stimulates the differentiation of xylem and phloem (phloem[+] cultures). Monoclonal antibodies specific for P-protein were identified by incubating free-hand stem sections of S. tortuosus in hybridoma supernatants, then in a goat anti-mouse antibody conjugated to fluorescein isothiocyanate (FITC), and observing the FITC under an epifluorescence microscope. Antibodies specific for P-protein in stem sections were used to probe nitrocellulose blots of polyacrylamide gels separating proteins isolated from both phloem(+) and phloem(-) tissue cultures. Immunoblots were incubated overnight in hybridoma supernatants followed by a secondary antibody conjugated to alkaline phosphatase. Three monoclonal antibodies-RS21, RS22, and RS23-bound to an 89-kD band in the phloem(+) lanes but failed to bind to any proteins in the phloem(-) lanes. In leaf sections of Arabidopsis thaliana processed by freeze-substitution, a mixture of RS21 and RS22 bound to the P-protein filaments in sieve elements, but not to any proteins in adjacent cells. A control antibody specific for tubulin did not bind to the P-protein filaments.