Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

Abstract

We have analysed the intracellular localisation of phytochrome in oat coleoptile cells by electron microscopy and confirm and extend light-microscopical findings of previous authors. We used indirect immuno-labeling with polyclonal antibodies against 60-KDa phytochrome from etiolated oat seedlings, and a gold-coupled second antibody, on ultrathin sections of LR-white-embedded material. In dark-grown seedlings, phytochrome-labeling is distributed diffusely throughout the cytoplasm. Organelles and membranes are not labeled. After photoconversion of the red-absorbing form of phytochrome to the far-red absorbing form (Pfr) (5-min red light; 660 nm), the label is sequestered uniquely in electron-dense areas within the cytoplasm. These areas are irregularly shaped, are often located in the vicinity of the vacuole, are not surrounded by a membrane, exclude cellular organelles and ribosomes and are not found in dark-grown material; an immediate 5-min farred light pulse after the red light does not cause these structures to disappear. After a dark period of 3-4 h following red-light irradiation, these electron-dense structures disappear together with any specific labeling. We suggest a Pfr-induced aggregation of an unknown, phytochrome-binding protein or proteins.