A partial characterization of the long-wavelength "Activated" far-red absorbing form of phytochrome

The spectral properties of Pfr in extracts prepared from red-irradiated oat tissue are similar to those for Pfr as measured in vivo, with a spectral peak at about 732 nm. The presence or absence of Mg(2+) or EDTA in the extraction medium is without effect on the spectral properties of the Pfr extracted from red-irradiated tissue. In contrast, the spectral properties of Pfr formed in vitro in extracts of non-irradiated oat tissue cleared of particulate matter are markedly different, the absorption peak being at about 725 nm. However, the red irradiation of the crude extract prior to its being cleared of particulate matter, results in the formation of a Pfr-species with spectral properties similar to Pfr as seen in vivo, suggesting the requirement for some particulate fraction for the formation of the long-wavelength Pfr form. The long-wavelength "activated" form of Pfr once extracted and partially purified is highly stable both at 0° and 25°C. Photoconversion of this long-wavelength form of Pfr to Pr initially results in the formation of a Pr species with a peak at 662 nm. In contrast, the peak location of Pr as isolated from non-irradiated tissue is at 667 nm.The 662 nm form of Pr is unstable and with time is transformed in a temperature-dependent reaction to a form with a peak at 667 nm. At 25°C this reaction is rapid, occurring in minutes, while at 0°-4°C it requires a few hours. At 25°C, immediate photoconversion of the 662 nm form of Pr back to Pfr results in the reformation of the long-wavelength form of Pfr. However, after being incubated for 15 min in the Pr form the peak shifts to 667 nm and the Pfr subsequently formed exhibits spectral characteristics identical to those of Pfr as formed in vitro, i.e., with a peak at 725 nm.