Spectral properties, membrane association, and stability of oat phytochrome.

The nature of the association of etiolated oat seedling phytochrome with a crude membranous fraction was investigated and correlated with the parallel changes in a phytochrome modulated physiological response. Firstly, the spectrophotometric assay of phytochrome in clear and turbid solutions was examined. Within given limits, in neither sample type was there an artifactual component of the phytochrome reading. The addition of CaCO3 particles was found to be capable of giving rise to artifacts and was therefore rejected. A strongly hydrophobic association of around 5% of total phytochrome with a pelletable fraction was observed using etiolated tissue Red light (R) in vivo increased this association to around 9%, an effect totally reversible by far-red light (FR) given immediately. The association appeared rather stronger for phytochrome pelleted after R than for "dark" or "R + FR" pelleted phytochrome. Supernatant and resuspended pellet phytochrome were found not to be significantly different in terms of their spectral characteristics or stability in vitro. Pfr disappeared in vivo more quickly from the pelletable fraction than from the supernatent fraction of seedlings given an increasing incubation in darkness after R. The amount of pelletable Pr remained relatively stable while supernatant Pr was lost. These observations suggested that supernatant Pfr was lost during the incubation, while pelletable Pfr was subject to both dark reversion and loss, although it was possible that supernatant Pr became pelletable. During the incubation, the percentage of pelletable phytochrome rose, and FR reversibility of the R-induced increase in phytochrome pelletability was lost, as was FR reversibility of the inhibition of etiolated oat coleoptile elongation induced by R. Both these losses in FR reversibility correlated with the change in total Pfr in supernatant, pellet and tissue samples, but with the change in the proportion of Pfr, only in the pellet sample. The significance of the observations is discussed with reference to the mode of action of phytochrome and a model is put forward to explain the observed characteristics of phytochrome association with the membranous pelletable fraction.