Clas Dahlin

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts

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°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.

The importance of the C-terminal region and Cys residues for the membrane association of the NADPH:protochlorophyllide oxidoreductase in pea.

In vitro chloroplast import reactions and thylakoid association reactions have been performed with a series of C‐terminal deletions and Cys‐to‐Ser substitution mutants of the pea NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99). C‐terminal deletions of the precursor POR (Δ362–400, Δ338–400, Δ315–400 and Δ300–400) were efficiently translocated across the chloroplast envelope. However, except the Δ396–400 mutant, no C‐terminal deletion mutants or Cys‐to‐Ser substitution (Cys119, Cys281 and Cys309) mutants resisted post‐treatment with thermolysin after the thylakoid association reactions. This suggests that these mutants were unable to properly associate to the thylakoids due to changes of the protein conformation of POR.

Characterization of the Plastid Import Reaction of the Pea Nadph: Protochlorophyllide Oxidoreductase (POR).

NADPH: protochlorophyllide (POR) is a vital enzyme in the biosynthesis of chlorophyll where it catalyzes the reduction of Pchlide into Chlide in a light-dependent manner. POR is nucleus-encoded and imported into the plastids where it is found at the inner membranes. Together with its substrate and the co-factor NADPH it forms a ternary complex which is needed for catalytical activity. The anomaly of a decreasing POR level during active chlorophyll synthesis was cleared with the discovery of two different POR proteins, POR-A and POR-B, in barley and Arabidopsis thaliana. During greening, POR-A is negatively regulated by light both at transcriptional and proteolytical levels. In addition, the import of POR-A, but not POR-B, has been suggested to require Pchlide in order to be translocated into the plastid [1]. In this respect, POR-A differs from other known nucleus-encoded plastid proteins, and as it appears, this requirements represents a novel and exclusive import characteristic.

In vitro integration of the ternary complex, POR-Pchlide-NADPH, into pea thylakoids

The nucleus-encoded NADPH: protochlorophyllide oxidoreductase (POR), catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) in higher plants. This is the first step in the biosynthetic pathway of chlorophyll that requires light. POR is mainly found in the prolamellar bodies (PLBs) in dark-grown plants but also at lower levels in greening plants. In barley and Arabidopsis thaliana two different PORs, POR-A and POR-B, have been found. POR-A has been suggested to be dependent upon the substrate, Pchlide, for translocation into the plastid [1]. After import, POR is assembled on the stromal side of the plastid inner membranes in accordance with the lack of transfer sequences for lumen translocation or membrane spanning regions. The catalytically active form is a ternary complex consisting of POR, Pchlide and NADPH. By analogy with the import reaction it could be speculated that Pchlide could play an active role in the interaction with the plastid inner membranes, e.g. is the integration facilitated by a pre-assembly of a ternary complex before the membrane integration? To address this question, we have used an in vitro integration assay to examine the requirement for ternary complex formation as a pre-requisite for membrane assembly of mature POR. We have also examined how mutations within POR affecting NADPH and Pchlide binding capacity (and thus ternary complex formation) affect integration. Our data suggest that it can not be excluded that a formation of a ternary complex in the stroma precede the integration reaction. However, the equally efficient integration of free POR, in the absence of exogenous NADPH and Pchlide, suggests that alternative membrane targeting pathways may exist.

Surface Charge Densities, Lipid Compositions and Fluidities of Thylakoid Membranes Showing Different Degrees of Stacking

Formation of thylakoid appressions occurs between regions of the membrane where the Coulombic repulsive forces between two adjacent membrane surfaces are decreased. This has been suggested to occur through lateral segregation of thylakoid components, such as displacement of negative surface charges to membrane regions not involved in stacking, and/or through cations screening negative membrane surface charges in the areas involved in stacking (1). The light-harvesting complex of photosystem II (LHC II) may partake in the stacking process (2) and phosphati-dylglycerol (PG) acylated with trans-hexadecenoic acid [16:1 (trans)] has been implicated to be involved in the organization of LHC II (3). Chloroplasts of plants grown with different concentrations of the herbicide SAN-9789 (Norflurazon, an inhibitor of carotenoid biosynthesis), contain thylakoids with different degrees of stacking. In this plant material the degree of thylakoid stacking is correlated with the amount of LHC II (4).