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

Abstract

The spectral properties of P_fr in extracts prepared from red-irradiated oat tissue are similar to those for P_fr as measured in vivo, with a spectral peak at about 732 nm. The presence or absence of Mg²⁺ or EDTA in the extraction medium is without effect on the spectral properties of the P_fr extracted from red-irradiated tissue. In contrast, the spectral properties of P_fr 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 P_fr-species with spectral properties similar to P_fr as seen in vivo, suggesting the requirement for some particulate fraction for the formation of the long-wavelength P_fr form. The long-wavelength “activated” form of P_fr once extracted and partially purified is highly stable both at 0° and 25°C. Photoconversion of this long-wavelength form of P_fr to P_r initially results in the formation of a P_r species with a peak at 662 nm. In contrast, the peak location of P_r as isolated from non-irradiated tissue is at 667 nm. The 662 nm form of P_r 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 P_r back to P_fr results in the reformation of the long-wavelength form of P_fr. However, after being incubated for 15 min in the P_r form the peak shifts to 667 nm and the P_fr subsequently formed exhibits spectral characteristics identical to those of P_fr as formed in vitro, i.e., with a peak at 725 nm.