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 chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple-exponential model at 14 degrees C and a double-exponential model at -170 degrees C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14 degrees C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different 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 Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating fight pulse, we found a single 5.0 ns component for Chlide-F688 (the Chlide-NADPH :protochlorophyllide oxidoreductase [POR]-NADP(+) complex) and an additional 1.6 ns component when the formation of Chlide-F696 (the Chlide-POR-NADPH complex) was promoted by exogenous NADPH, From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide-F696 as being 70%.