Analysis of fluorescence lifetime of protochlorophyllide and chlorophyllide in isolated etioplast membranes measured from multifrequency cross-correlation phase fluorometry

The fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyll ide (Chlide) in wheat etioplast membranes were analyzed using a multiexpone ntial fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple-exponential model at 14 degrees C an d a double-exponential model at -170 degrees C were adequate to describe th e Pchlide fluorescence decay. We discuss the origin of the three fluorescen ce lifetime components at 14 degrees C on the basis of the dependence of th eir fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of diff erent Pchlide forms in steady-state fluorescence spectra, The fluorescence decay of the main Pchlide form, photoactive Pchlide-F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5-6.0 ns are ascribed to the second photoactive form, Pchlide-F645, and to nonphotoactive Pchlid e forms, respectively. In etioplast membranes preilluminated by a short sat urating fight pulse, we found a single 5.0 ns component for Chlide-F688 (th e Chlide-NADPH :protochlorophyllide oxidoreductase [POR]-NADP(+) complex) a nd an additional 1.6 ns component when the formation of Chlide-F696 (the Ch lide-POR-NADPH complex) was promoted by exogenous NADPH, From the fluoresce nce lifetime results we evaluated the quantum yield of the primary photorea ction by Chlide-F696 as being 70%.