Activation parameters of the blue shift (Shibata shift) subsequent to protochlorophyllide phototransformation.
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The Shibata shift was analyzed in flash irradiated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates in the pressure range of 0.1 to 500 MPa, at temperatures of 20, 30 and 40 degrees C. The kinetics of the blue shift (called Shibata shift in case of intact leaves) was followed by repeated recording of fluorescence emission spectra after phototransformation. At 20 degrees C, above 100 MPa, the blue shift slowed down remarkably. Two components of the blue shift could be distinguished, one was pressure-dependent and the other was almost pressure-independent. The pressure-independent component can be associated with minor conformational changes of the NADPH:protochlorophyllide oxidoreductase (POR) enzyme, followed by molecular movements of the newly formed chlorophyllide molecules. The calculated activation volume of the pressure-dependent component was 43+/-11 cm(3) mol(-1) at 20 degrees C. This value reflects major molecular reorganizations in the lipid system of the membrane and in the chlorophyllide-protein complexes, and corresponds to changes of the tertiary structure of proteins which can proceed directly or indirectly via structural changes of the membrane lipids. The process was inhibited by 300 and 400 MPa at 30 and 40 degrees C, respectively. The activation volume reduced to 35+/-1.5 cm(3) mol(-1) at 40 degrees C. The decrease of the activation volume with increasing temperature indicates that the blue shift requires loosened lipid structures. The activation energy of the blue shift (measured between 10 and 40 degrees C at atmospheric pressure) was 100+/-20 kJ/mol, indicating that the structural change involves rearrangement of strong molecular interactions. (+info)
Chloroplast biogenesis 88. Protochlorophyllide b occurs in green but not in etiolated plants.
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It has recently been reported that protochlorophyllide (Pchlide) b is an abundant pigment in barley etioplasts but is rather unstable, as it is rapidly converted to Pchlide a by 7-formyl reductase during pigment extraction with conventional 80% acetone (Reinbothe, S., Pollmann, S., and Reinbothe, C. (2003) J. Biol. Chem. 278, 800-806). It has also been claimed that extraction of barley etioplasts with 100% acetone containing 0.1% diethyl pyrocarbonate prevents the conversion of Pchlide b to Pchlide a and leads to the detection of large amounts of Pchlide b in the isolated etioplasts. In this work the extraction protocol of Reinbothe et al. is compared with the more conventional 80% aqueous acetone extraction method. No Pchlide b was detected either in etiolated barley leaves or isolated barley etioplasts irrespective of the extraction protocol. On the other hands, small amounts of Pchlide b were detected in green barley leaves and isolated chloroplasts, extracted either with 80% acetone or 100% acetone containing 0.1% diethyl pyrocarbonate. It is concluded that the proposed occurrence of a light-harvesting POR-Pchlide-a,b complex in etiolated plant tissues is untenable, and its ensuing consequences and implications, for the greening process, are irrelevant. (+info)
Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide.
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CHL27, the Arabidopsis homologue to Chlamydomonas Crd1, a plastid-localized putative diiron protein, is required for the synthesis of protochlorophyllide and therefore is a candidate subunit of the aerobic cyclase in chlorophyll biosynthesis. delta-Aminolevulinic acid-fed antisense Arabidopsis plants with reduced amounts of Crd1/CHL27 accumulate Mg-protoporphyrin IX monomethyl ester, the substrate of the cyclase reaction. Mutant plants have chlorotic leaves with reduced abundance of all chlorophyll proteins. Fractionation of Arabidopsis chloroplast membranes shows that Crd1/CHL27 is equally distributed on a membrane-weight basis in the thylakoid and inner-envelope membranes. (+info)
Substrate-dependent and organ-specific chloroplast protein import in planta.
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The NADPH-dependent protochlorophyllide (Pchlide) oxidoreductase (POR) is unique because it is a photoenzyme that requires light for its catalytic activity and uses Pchlide itself as a photoreceptor. In Arabidopsis, there are three structurally related PORs, denoted PORA, PORB, and PORC. The import of one of them, PORA, into plastids of cotyledons is substrate dependent. This substrate dependence is demonstrated in intact seedlings of wild-type Arabidopsis and two mutants, xantha2, which is devoid of Pchlide, and flu, which upon redarkening rapidly accumulates Pchlide. In true leaves, PORA uptake does not require the presence of Pchlide. The organ specificity of the substrate-dependent import of PORA reveals a means of controlling plastid protein translocation that is closely associated with a key step in plant development, the light-dependent transformation of cotyledons from a storage organ to a photosynthetically active leaf. (+info)
Identification of plastid envelope proteins required for import of protochlorophyllide oxidoreductase A into the chloroplast of barley.
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Chloroplasts synthesize an abundance of different tetrapyrrole compounds. Among them are chlorophyll and its precursor protochlorophyllide (Pchlide), which accumulate in light- and dark-grown plants, respectively. Pchlide is converted to chlorophyllide by virtue of the NADPH:Pchlide oxidoreductase (POR), which, in angiosperms, is the only known light-dependent enzyme of the chlorophyll biosynthetic pathway. In etiolated barley plants, two closely related POR proteins exist termed PORA and PORB, which are nuclear gene products. Here we identified plastid envelope proteins that interact with the cytosolic PORA precursor (pPORA) during its posttranslational chloroplast import. We demonstrate that pPORA interacts with several previously unreported components. Among them is a Pchlide a oxygenase, which provides Pchlide b as import substrate for pPORA, and a tyrosine aminotransferase thought to be involved in the synthesis of photoprotective vitamin E. Two other constituents were found to be orthologs of the GTP-binding proteins Toc33/34 and of the outer plastid envelope protein Oep16. (+info)
Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley.
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A photoactive protochlorophyllide-protein complex with absorbance and fluorescence maxima at 648 and 653 nm was detected in greening barley leaves without any re-darkening. The variations of the amplitudes of the absorbance and the fluorescence of the photoactive protochlorophyllide with greening time at two different light intensities indicate a close relationship between the rate of chlorophyll synthesis and the amount of the complex during the first hours. The chlorophyllide resulting from photoreduction during greening has an absorbance maximum at 684 nm, which shifts towards a shorter wavelength within a few seconds, indicating rapid liberation of the pigment from the enzyme. We conclude that chlorophyll accumulation proceeds through continuous regeneration and phototransformation of the photoactive complex. (+info)
Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus species.
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Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet. In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis. Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR. DVR is encoded by a single copy gene in the A. thaliana genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of A. thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b. Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls. A DVR homolog was not found in the genome of P. marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus. (+info)
Organization of protochlorophyllide oxidoreductase in prolamellar bodies isolated from etiolated carotenoid-deficient wheat leaves as revealed by fluorescence probes.
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Carotenoid importance for membrane organization of NADPH protochlorophyllide oxidoreductase (POR) was studied by comparing interaction of two membrane fluorescent probes with proteins in prolamellar bodies isolated from norflurazon-treated wheat plants (cdPLBs) to those isolated form plants with normal carotenoid amount (oPLBs). The tryptophan fluorescence quenching by 1-anilino-8-naphthalene sulfonate (attached to the surface of membrane lipid phase) and pyrene (situated deep into the fatty acid region of membrane lipids) was used to locate the position of POR molecules toward lipid phase, to analyze their supramolecular organization and the light-induced structural transitions. Our results showed that the pigment-protein complexes of cdPLBs were larger than those of oPLBs. Upon flash irradiation the aggregates of both types of PLB dissociated into smaller units but in cdPLBs this process was accompanied by reorientation of the POR molecules closer to the lipid surface and/or dissociation from the lipids. These results revealed that carotenoid deficiency led to a looser attachment of POR to the lipid phase and its early (in comparison with oPLBs) dissociation from the membranes during the light-induced transformation of cdPLBs. This might be one of the reasons for the inability of carotenoid-deficient plants to form functional plastids. (+info)