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

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Abstract
The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), NADPH:Pchlide oxidoreductase (POR; EC 1.6.99.1), is a nuclear‐encoded protein that is post‐translationally imported to the plastid. In barley and Arabidopsis thaliana, the reduction of Pchlide is controlled by two different PORs, PORA and PORB. To characterise the possible Pchlide dependency for the import reaction, radiolabelled precursor proteins of barley PORA and PORB (pPORA and pPORB, respectively) were used for in vitro assays with isolated plastids of barley and pea with different contents of Pchlide. To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley plants were grown in darkness or in greenhouse conditions for 6 days. Alternatively, greenhouse‐grown pea plants were incubated for 4 days in darkness before plastid isolation, or chloroplasts isolated from greenhouse‐grown plants were incubated with Δ‐aminolevulinic acid (ALA), an early precursor in the Chl biosynthesis resulting in elevated Pchlide contents in the plastids. Both barley pPORA and pPORB were effectively imported into barley and pea chloroplasts isolated from the differentially 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‐enriched fractions from chloroplasts and etioplasts of barley indicated that no post‐import degradation of the proteins occurred in the stroma, irrespective of whether the incubation was performed in darkness or in light.