Long-lived Intermediates in Phytochrome

Irradiation of phytochrome solutionsewith a high-intensity mixed red and far red light source causes measurable absorbancy increases at 543 nm. Evidence is presented that these absorbancy increases are caused by accumulation of intermediates on the PR to PFR pathway with relatively slow thermal decay constants. Kinetic analysis of the decay signals is consistent with the interpretation that the signals represent simultaneous independent and parallel decay of 2 species by first order kinetics to PFm. If actinic light intensity is kept constant and exposure time changed, the relative amounts of the 2 components change, with proportionately more of the rapidly decaying species present following short exposure times. If the amount of the intermediates is decreased by decreasing actinic light intensity at constant exposure time, however, the relative amounts of the 2 remain constant. The Ql0 for inter. mediate decay following illumination is approximately 2.0, while that for complete phototransformation of the pigment in either direction is very close to 1.0. Incomplete transformation of PI to PFit, caused by overlapping absorption of the 2 forms, is shown by the presence of intermediates (indicating cycling of the pigment) in continuous red light. Such intermediates do not appear in oontinuous far red, indicating a rate of pigment cycling below detection by the availablo instrumentation. The elegant flash photolysis experiments by Linschitz and his co-workers have revealed several fundamental aspects of phototransformation of the pigment phytochrome (9, 10). First, transformation in both directions involves the formation and decay of a number of spectrally distinguishable intermediates. Second, the Pit to Pint pathway involves different intermediates than the reverse photoreaction. Third, concurrent parallel dark reactions lead to the formation of the final product in both directions. 'Supported by National Science Foundation Grants GB-2846 and GB-6633 and a grant from Research Corporation to W. R. Briggs. 2 Most of the experiments described in this paper were performed while the senior author was a member of the Department of Biological Sciences, Stanford UTniversity, Stanford, California, 94305. Finally, at least 2 intermediates on the Pn to PFI pathway have rate constants for decay to PFit at least 10-fold smaller than the rate constants for anv of the other intermediates on either pathway. Low temperature studies by Pratt and Butler (12) -and by Cross et al. (7) suggest that an early event in PR transformation is the formation of an intermediate which can be photoreversibly driven back to PR, but there is no evidence that more than the initial photoreaction in transformation of PR to PFII in a forward direction is light-driven. Spruit has also observed phytochrome intermediates at liquid nitrogen temperature (14-17) but interpretations of his data are complicated by possible interference in the spectral measurements by protochlorophyll, chlorophyll, or both. It seemed possible that irradiations of phytochrome solutions with a high intensity mixture of