Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.
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The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide. (+info)
Altered monovinyl and divinyl protochlorophyllide pools in bchJ mutants of Rhodobacter capsulatus. Possible monovinyl substrate discrimination of light-independent protochlorophyllide reductase.
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In land plants in particular, it has been well established that chlorophyll intermediates, Mg-protoporphyrin, Mg-protoporphyrin monomethylester, protochlorophyllide, and chlorophyllide occur as monovinyl and divinyl forms. The pool of monovinyl and divinyl intermediates differ according to species, age of tissue, and light regime. In this study, we investigated the monovinyl and divinyl characteristics of protochlorophyllide and chlorophyllide in the purple non-sulfur photosynthetic eubacterium Rhodobacter capsulatus. Our results indicate that mutations in genes known to completely block the reduction of protochlorophyllide to chlorophyllide (such as bchN, bchB, and bchL mutants), accumulate a pool of monovinyl and divinyl forms of protochlorophyllide just as observed in plants. However, we also observed that directed insertion and deletion mutations in bchJ, a gene located in the photosynthesis gene cluster, affected the ratio of monovinyl and divinyl protochlorophyllide. Specifically, bchJ-disrupted strains accumulate reduced levels of bacteriochlorophyll concomitant with the accumulation of divinyl protochlorophyllide. Mutants of bchJ in combination with a second mutation in bchL still produce a mixed pool of monovinyl and divinyl protochlorophyllide; however, the ratio of monovinyl to divinyl protochlorophyllide is skewed in favor of divinyl protochlorophyllide. These results thus identify bchJ as the first sequenced gene that affects the divinyl to monovinyl ratio of photopigment intermediates in any photosynthetic organism. In addition, the results of our study also suggest that light-independent protochlorophyllide reductase is discriminatory for a monovinyl substrate. (+info)
8-vinyl reduction and chlorophyll a biosynthesis in higher plants.
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A technique involving solid-phase extractions and polyethylene h.p.l.c. suitable for the routine compositional analysis of the total protochlorophyllide pool of plants is described. The resynthesis kinetics of the individual components of the pool have been studied in briefly illuminated etiolated tissue of wheat (Triticum aestivum) and cucumber (Cucumis sativus) during subsequent redarkening. The data are interpreted in terms of a precursor-product relationship between the di- and mono-vinyl analogues of protochlorophyllide during their reaccumulation in darkness. The interconversion is assumed to be catalysed by an 8-vinyl reductase, which shows greater activity in wheat than in cucumber. Analyses of the redox state of the nicotinamide nucleotide of the pool during the process are compatible with NADPH as the cofactor of the putative reductase. (+info)
Chloroplast biogenesis. Net synthesis of protochlorophyllide from magnesium-protoporphyrin monoester by developing chloroplasts.
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Developing chloroplasts were isolated from greening Cucumis cotyledons in a cofactor-enriched medium and were incubated in the dark with 14C-labeled and unlabeled magnesium-protoporphyrin monoester. The metabolic pools between protoporphyrin and protochlorophyllide were monitored spectrofluorometrically. The incorporation of the 14C label into protochlorophyllide was also determined. It was shown that magnesium-protoporphyrin monoester, a postulated intermediate of the chlorphyll biosynthetic pathway, was convertible into protochlorophyllide with relatively high yields. Since protochlorophyllide is the immediate precursor of chlorophyll a it was concluded that magnesium-protoporphyrin monoester was indeed an intermediate of the chlorophyll biosynthetic pathway. (+info)
The regulation of enzymes involved in chlorophyll biosynthesis.
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All living organisms contain tetrapyrroles. In plants, chlorophyll (chlorophyll a plus chlorophyll b) is the most abundant and probably most important tetrapyrrole. It is involved in light absorption and energy transduction during photosynthesis. Chlorophyll is synthesized from the intact carbon skeleton of glutamate via the C5 pathway. This pathway takes place in the chloroplast. It is the aim of this review to summarize the current knowledge on the biochemistry and molecular biology of the C5-pathway enzymes, their regulated expression in response to light, and the impact of chlorophyll biosynthesis on chloroplast development. Particular emphasis will be placed on the key regulatory steps of chlorophyll biosynthesis in higher plants, such as 5-aminolevulinic acid formation, the production of Mg(2+)-protoporphyrin IX, and light-dependent protochlorophyllide reduction. (+info)
The light intensity dependence of protochlorophyllide photoconversion and its significance to the catalytic mechanism of protochlorophyllide reductase.
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The light-dependent step in chlorophyll synthesis by higher plants involves hydrogen transfer from NADPH+ to the porphyrin protochlorophyllide catalysed by the photoenzyme protochlorophyllide reductase. The light intensity dependence of the process has been studied in vitro using wheat etioplast membranes. The results suggest that a single photochemical event is involved in the photoconversion. In support of this conclusion we also demonstrate that illumination of these membranes with a train of ultrashort (150 fs) flashes resulted in chlorophyll accumulation. The significance of the findings in terms of possible mechanisms for the reductase are discussed. (+info)
Chloroplast biogenesis. Net synthesis of protochlorophyllide from protoporphyrin IX by developing chloroplasts.
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Developing chloroplasts were isolated from greening Cucumis cotyledons in a co-factor-enriched medium and were incubated in the dark with 14C-labeled and unlabeled protoporphyrin IX. The metabolic pools between protoporphyrin and protochlorophyllide were monitored spectrofluorometrically. The incorporation of the 14C label into protochlorophyllide was also determined. It was shown that protoporphyrin IX, a postulated intermediate of the chlorophyll biosynthetic pathway, was convertible into protochlorophyllide. Since protochlorophyllide is the immediate precursor of chlorophyll a it was concluded that protoporphyrin IX was indeed an intermediate of the chlorophyll biosynthetic pathway. (+info)
Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.
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The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655. (+info)