Immunofluorescence visualization of phytochrome in Pisum sativum L. epicotyls using monoclonal antibodies

We have investigated the cellular distribution of phytochrome in epicotyls of dark-grown pea (Pisum sativum L.) seedlings using monoclonal antibodies to pea phytochrome. Screening of the eight available antibodies both by an enzymelinked immunosorbent assay (ELISA) and by their ability to visualize phytochrome in situ by immunocytochemical fluorescence demonstrated that: (1) three antibodies work well for immunofluorescence; (2) none of the eight antibodies discriminates between the red- and the far-red-absorbing forms of phytochrome (Pr, Pfr) as assayed by ELISA; (3) the antigenicity of phytochrome is reduced by fixation with formaldehyde with respect to all eight antibodies; and (4) two antibodies that bind well to formaldehyde-fixed phytochrome as assayed by ELISA do not bind well to phytochrome in situ. Phytochrome is observed in both cortical and stomatal guard cells of the epicotyl and exhibits a homogeneous cytoplasmic distribution in non-irradiated tissue. After red-light (R) treatment phytochrome becomes transiently inaccessible to antibodies. If maintained in the Pfr form for 10 min at room temperature before fixation, at least a portion of the phytochrome pool becomes accessible to antibodies and assumes a "sequestered" distribution. Both of these effects are almost entirely either prevented or reversed by subsequent far-red light treatment. We believe that the transient inaccessibility of phytochrome to antibodies after R irradiation is not a function of its conformational state. We suggest instead that R treatment rapidly induces an association of phytochrome with a subcellular component that interferes with antibody binding and that the "sequestered" areas represent a phytochrome pool that is distinct from both the diffusely distributed phytochrome in non-irradiated cells and from that phytochrome which is inaccessible to antibodies immediately after R irradiation.