INTRACELLULAR PHYTOCHROME DISTRIBUTION AS A FUNCTION OF ITS MOLECULAR FORM AND OF ITS DESTRUCTION

The immunocytochemically observed intracellular redistribution of phytochrome as a function of its molecular form is described by utilizing color photomicrography. The reversible change from a diffuse to a discretely localized distribution following photoconversion of the red-absorbing Pr form to the far-red-absorbing Pfr form observed with etiolated oat (Avena sativa L., cv. Garry) coleoptile parenchyma cells is not seen with etiolated wheat (Triticum sativum L., cv. unknown), barley (Hordeum vulgare L., cv. Harrison), or rye (Secale cereale L., cv. Balbo). Whether redistribution in these latter cases does not occur or is below the limit of detection is not known. Upon continuous actinic irradiation, phytochrome, which is discretely localized as Pfr, rapidly disappears by both immunocytochemical and spectral assay. However, after about 90 min irradiation, a new association of phytochrome with nuclei is evident which is more pronounced after 4 or 8 h of irradiation. With longer irradiation times there is a total loss of antigenically detectable phytochrome at the resolution employed in these experiments. PHYTOCHROME DISTRIBUTION, both within tissues (Pratt and Coleman, 1971, 1974) and within cells (Coleman and Pratt, 1974a,b; Mackenzie et al., 1975) has been described by an indirect immunocytochemical assay. While Pr, the morphogenically inactive red-absorbing form of the chromoprotein, has a diffuse distribution within most cells examined, Mackenzie et al. (1975) have shown with oat and rice shoots that following phototransformation of Pr to Pfr, the morphogenically active far-red-absorbing form of the pigment, phytochrome becomes discretely localized or sequestered. If Pfr is photoconverted back to Pr by far-red light, and if the tissue is subsequently left in darkness, phytochrome slowly returns to the diffuse distribution originally seen in dark-grown control tissue. Immunocytochemistry of phytochrome has also been employed as a tool for following Pfr destruction (Coleman and Pratt, 1974c; Pratt, Kidd, and Coleman, 1974). Although these authors demonstrated that antigenically detectable phytochrome was lost in parallel to the loss of I Received for publication 15 December 1977; revision accepted 3 April 1978. 4 Present address: Department of Pharmacology, Stanford University Medical Center, Stanford, California 94305. This research was supported by National Science Foundation grants GB-17057 and PCM75-19125 to L. H. P. J. M. M. was supported in part by National Institutes of Health Training Grant TOIGM0036 and by the Department of Plant Biology, Carnegie Institution of Washington, Stanford, California. C.I.W.-D.P.B. Publication No. 621. spectrally detectable phytochrome, they were unaware that photoconversion of Pfr back to Pr, which they employed as a method for stopping the destruction reaction, might produce changes in distribution during subsequent fixation at room temperature (Mackenzie et al., 1975). Thus, information concerning the intracellular distribution of phytochrome during destruction cannot be obtained from these experiments. The purpose of this paper is to present a further examination of phytochrome distribution within the cell. Color photography is employed to present new information about these movements as well as to follow carefully the intracellular distribution of phytochrome during continuous illumination by light. MATERIALS AND METHODS-Oat (Avena sativa L., cv. Garry), rye (Secale cereale L., cv. Balbo), barley (Hordeum vulgare L., cv. Harrison), and wheat (Triticum sativum L., cv. unknown) grains were sown on moist cellulose packing material and grown in open trays for four days in darkness at 25 C (Pratt, 1973). Except during spectral assay or until completion of fixation, all handling was under dim, green safelights. Photoreversibility measurements were made with a custom-built dual wavelength (667 and 723 nm) spectrophotometer (Kidd and Pratt, 1973). Actinic red and far-red sources are the same as used previously (Kidd and Pratt, 1973; Mackenzie et al., 1975).