Photoactive protochlorophyllide regeneration in cotyledons and leaves from higher plants

Chlorophyll accumulation during greening implies the continuous transformation of photoactive protochlorophyllide (Pchlide) to chlorophyllide. Since this reaction is a light-dependent step, the study of regeneration of photoactive Pchlide under a continuous illumination is difficult. Therefore this process is best studied on etiolated plants during a period of darkness following the initial photoreduction of photoactive Pchlide. In this study, the regeneration process has been studied using spinach cotyledons, as well as barley and bean leaves, illuminated by a single saturating flash. The regeneration was characterized using 77 K fluorescence emission and excitation spectra and high-performance liquid chromatography. The fluorescence data indicated that the same spectral forms of photoactive Pchlide are regenerated by different pathways: (1) photoactive Pchlide regeneration starts immediately after the photoreduction through the formation of a nonphotoactive Pchlide form, emitting fluorescence at approximately 651 nm. This form is similar to the large aggregate of photoactive Pchlide present before the illumination, but it contains oxidized form of nicotinamide adenine dinucleotide phosphate, instead of the reduced form (NADPH), in the ternary complexes; and (2) after the dislocation of the large aggregates of chlorophyllide-light-dependent NADPH:Pchlide a photooxidoreductase-NADPH ternary complexes, the regeneration occurs at the expense of the several nonphotoactive Pchlide spectral forms present before the illumination.