Protochlorophyllide-independent import of two NADPH : Pchlide oxidoreductase proteins (PORA and PORB) from barley into isolated plastids

The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chl orophyllide (Chlide), NADPH:Pchlide oxidoreductase (POR; EC 1.6.99.1), is a nuclear-encoded protein that is post-translationally imported to the plast id, In barley and Arabidopsis thaliana, the reduction of Pchlide is control led by two different PORs, PORA and PORE. To characterise the possible Pchl ide dependency for the import reaction, radiolabelled precursor proteins of barley PORA and PORE (pPORA and pPORB, respectively) were used for in vitr o assays with isolated plastids of barley and pea with different contents o f Pchlide, To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley plants were grown in darkness or in green house conditions for 6 days. Alternatively, greenhouse-grown pea plants wer e incubated for 4 days in darkness before plastid isolation, or chloroplast s isolated from greenhouse-grown plants were incubated with delta-aminolevu linic acid (ALA), an early precursor in the Chi biosynthesis resulting in e levated Pchlide contents in the plastids, Both barley pPORA and pPORB were effectively imported into barley and pea chloroplasts isolated from the dif ferentially treated plants, including those isolated from greenhouse-grown plants. The absence or presence of Pchlide did not significantly affect the import capacity of barley pPORA or pPORB, Assays performed on stroma-enric hed fractions from chloroplasts and etioplasts of barley indicated that no post-import degradation of the proteins occurred in the stroma, irrespectiv e of whether the incubation was performed in darkness or in light.