Protochlorophyllide-NADP(+) and protochlorophyllide-NADPH complexes and their regeneration after flash illumination in leaves and etioplast membranesof dark-grown wheat

The fast (1 min) regeneration process of the photoactive Pchlide forms afte r a light flash was studied in etiolated wheat leaves, and this process was simulated in vitro by incubating etioplast inner membranes of wheat with e xcess NADPH or NADP(+). The 77 K fluorescence spectra were recorded after f lash illumination, dark incubation and a subsequent flash illumination of t he samples. A non-photoactive Pchlide form with an emission maximum at 650 nm was transiently detected in leaves during regeneration of a photoactive Pchlide form with an emission maximum at 654 nm. Gaussian deconvolution of fluorescence spectra of isolated membranes showed that this 650 nm form app eared in conditions of excess NADP(+), as suggested in previous studies. Ad ditionally a Pchlide form emitting at 638.5 nm was detected in the same con ditions. The analysis of the spectra of leaves at different times after a f lash indicated that these two non-photoactive forms are involved as interme diates in the regeneration of photoactive Pchlide. This regeneration is in correlation with the production of the Chlide form emitting at 676 nm. The results demonstrate that, in vivo, part of the NADPH:protochlorophyllide ox idoreductase is reloading with nonphotoactive Pchlide on a fast time-scale and that the 676 nm Chlide form is the released product of the phototransfo rmation in this process.