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

Abstract

bated for 4 days in darkness before plastid isolation, or The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), NADPH:Pchlide oxi-chloroplasts isolated from greenhouse-grown plants were incubated with -aminolevulinic acid (ALA), an early precursor in doreductase (POR; EC 1.6.99.1), is a nuclear-encoded protein that is post-translationally imported to the plastid. In barley the Chl biosynthesis resulting in elevated Pchlide contents in the plastids. Both barley pPORA and pPORB were effectively and Arabidopsis thaliana, the reduction of Pchlide is conimported into barley and pea chloroplasts isolated from the trolled by two different PORs, PORA and PORB. To characterise the possible Pchlide dependency for the import reaction, differentially treated plants, including those isolated from greenhouse-grown plants. The absence or presence of Pchlide radiolabelled precursor proteins of barley PORA and PORB did not significantly affect the import capacity of barley (pPORA and pPORB, respectively) were used for in vitro assays with isolated plastids of barley and pea with different pPORA or pPORB. Assays performed on stroma-enriched fractions from chloroplasts and etioplasts of barley indicated contents of Pchlide. To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley that no post-import degradation of the proteins occurred in the plants were grown in darkness or in greenhouse conditions for stroma, irrespective of whether the incubation was performed in darkness or in light. 6 days. Alternatively, greenhouse-grown pea plants were incu-

A substrate-independent, 14:3:3 protein-mediated plastid import pathway of NADPH:protochlorophyllide oxidoreductase A

“…For 35 S-pFd and 35 S-pSSU only two different precursor populations were found of which the faster eluting population had an elution time between fractions A and B containing the 35 SpPORA and 35 S-pPORB (Fig. 2B, fractions [20][21][22][23][24]. Western blotting using a heterologous anti-Hsp70 antiserum of tomato identified a 70-kDa band, which was most abundant in fractions 4-20 (Fig.…”

“…At this stage of development, the newborn seedlings prepare for light harvesting and the switch from heterotrophic to photoautotrohic growth. In older plants, the pathway remains active as well, but its operation is superimposed by a second pathway that requires binding of 14:3:3 proteins and Hsp70 and can explain the previously observed substrate-independent import mode of pPORA (21,22,32). The role of this pathway could be to provide a back-up system for the sequestration of free pigment in a nonhazardous form.…”

Chloroplast biogenesis: The use of mutants to study the etioplast–chloroplast transition

“…Whereas prePORB seems to follow a general import pathway, the import of prePORA into isolated chloroplasts or etioplasts was suggested to depend on its substrate Pchlide (14,15,17). Although these results could not be verified in vitro (23,24), in vivo data demonstrated that prePORA requires its substrate Pchlide to become imported into chloroplasts of cotyledons (12,13). The substrate dependence is lost in vivo in true leaves and therefore seems developmentally regulated (12).…”

The outer plastid envelope protein Oep16: Role as precursor translocase in import of protochlorophyllide oxidoreductase A

“…Work of Dahlin, Aronsson, and colleagues questioned the operation of the substrate-dependent import pathway (19)(20)(21). However, Kim and Apel (22) demonstrated substrate-dependent import of pPORA in vivo with Arabidopsis thaliana transformed with chimeric DNA encoding fusions of the transit sequence of pPORA or the whole pPORA precursor polypeptide and GFP.…”