Analysis of Fluorescence Lifetime of Protochlorophyllide and Chlorophyllide in Isolated Etioplast Membranes Measured from Multifrequency Cross‐correlation Phase Fluorometry

Abstract

AbstractThe 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°C and a double‐exponential model at — 170°C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14°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 light pulse, we found a single 5.0 ns component for Chlide‐F688 (the Chlide‐NADPH: protochlorophyllide oxidoreductase [PORJ‐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%.