With chlorophyll pigments from prolamellar bodies to light-harvesting complexesChrister Sundqvist and Clas Dahlin Sundqvist, C. and Dahlin, C, 1997. With chlorophyll pigments from prolamellar bodies to light-harvesting complexes. -Physiol. Plant 100: 748-759.The biosynthetic chain leading from 5-aminolevulinic acid to chlorophyll is localised to the plastid. Many of the enzymes are nuclear-encoded. NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) is one such enzyme which is encoded by two different genes and can exist in an A and a B form. Its import into the plastid seems to be facilitated when protochlorophyllide is present in the chloroplast envelope. Within the plastid the reductase is assembled to thylakoids or prolamellar bodies. The specific properties of the reductase together with the specific properties of the lipids present in the etioplast inner membranes promote the formation of the three-dimensional regular network of the prolamellar bodies. The reductase forms a temary complex with protochlorophyllide and NADPH that gives rise to different spectral forms of protochlorophyllide. Light transforms protochlorophyllide into chlorophyllide and this photoreaction induces a conformational change in the reductase protein which leads to a process of disaggregation of enzyme, pigment aggregates and membranes, which can be followed spectroscopically and with electron microscopy. The newly formed chlorophyllide is esterified by a membrane-bound nuclear-encoded chlorophyll synthase and the chlorophyll molecule is then associated with proteins into active pigment protein complexes in the photosynthetic machinery.

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.

Plastid development involves the programmed accumulation of proteins. Most plastid proteins are synthesized in the cytosol and imported into the organelle by an envelope-based protein import apparatus. Previous studies have shown that developmental rates of protein accumulation correspond to mRNA levels. Here, we examined the relationship between plastid development and the activity of the protein import apparatus. Developing plastids, primarily from wheat leaves, were analyzed for their protein import capability in vitro. Import capability, initially high in proplastids, declined as much as 20-fold as plastid development approached either the mature etioplast or the mature chloroplast. The observed decline was not due to senescence, nonspecific inhibitors, or protein turnover. Furthermore, the import capability of mature etioplasts, initially very low, was transiently reactivated during light-mediated redifferentiation into chloroplasts. These results suggest that plant cells regulate the import apparatus in concert with the protein demands of the developing plastids.

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-

Plastid development involves the programmed accumulation of proteins. Most plastid proteins are synthesized in the cytosol and imported into the organelle by an envelope-based protein import apparatus. Previous studies have shown that developmental rates of protein accumylation correspond to mRNA levels. Here, we examined the relationship between plastid development and the activity of the protein import apparatus. Developing plastids, primarily from wheat leaves, were analyzed for their protein import capability in vitro. lmport capability, initially high in proplastids, declined as much as 20-fold as plastid development approached either the mature etioplast or the mature chloroplast. The observed decline was not due to senescence, nonspecific inhibitors, or protein turnover. Furthermore, the import capability of mature etioplasts, initially very low, was transiently reactivated during lightmediated redifferentiation into chloroplasts. These results suggest that plant cells regulate the import apparatus in concert with the protein demands of the developing plastids.