Prolamellar Bodies Of Etioplasts Research Articles

Para-crystalline membrane structures resembling prolamellar bodies in the invasion zones of indeterminate root nodules of Vicia faba L.

Novel para-crystalline structures resembling prolamellar bodies in etioplasts were found in the invasion zones of indeterminate root nodules of Vicia faba, which possess persistent meristems and exhibit sequential developmental stages. The para-crystalline structures existed in most cells in the area of the invasion zone and a hexagonal arrangement of tubular membranes was recognized. Extensive membranes, apparently procured from the structures, were often in contact with the bacteria in young infected cells. We propose that the para-crystalline structures serve as a reservoir of membranes for the formation of the numerous symbiosomes that propagate and fill the infected cells, and suggest naming them pro-symbiosome membrane bodies.

Putative Mutation Mechanism and Light Responses of a Protochlorophyllide Oxidoreductase-Less Barley Mutant NYB

NYB (Nanchong Yellow Barley) is a Chl-less barley mutant, which is controlled by a recessive nuclear gene. It is the only protochlorophyllide oxidoreductases (POR)-less barley mutant known in the world. The putative mechanism of the mutation and its Chl synthesis and plastid development are studied here. Neither PORC nor an additional copy of porB could be detected in barley. porB mRNAs are normally expressed and correctly spliced in the mutant. However, the import of PORA, PORB, LHCIIb1 (light harvesting complex II b1) and SSU (small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) proteins to the plastid was greatly hampered in the mutant. We presume that a common translocon is mutated in NYB. The content of the supramolecular light-harvesting POR complex LHPP (light-harvesting NADPH:protochlorophyllide oxidoreductase:protochlorophyllide) and the density of prolamellar bodies in etioplasts are decreased in the mutant. However, no further oxidative damage could be observed for the de-etiolated mutant seedlings after a dark to light shift. Development of the plastid is arrested (less stacking) in NYB, and the mutant becomes more yellowish in high-light conditions, with dwarfing of seedlings and decreased yield. The physiological significance and developmental roles of POR proteins and LHPP in barley cells are discussed.

Arabidopsis protochlorophyllide oxidoreductase A (PORA) restores bulk chlorophyll synthesis and normal development to a porB porC double mutant

In angiosperms the strictly light-dependent reduction of protochlorophyllide to chlorophyllide is catalyzed by NADPH:protochlorophyllide oxidoreductase (POR). The Arabidopsis thaliana genome encodes three structurally related but differentially regulated POR genes, PORA, PORB and PORC. PORA is expressed primarily early in development-during etiolation, germination and greening. In contrast, PORB and PORC are not only expressed during seedling development but also throughout the later life of the plant, during which they are responsible for bulk chlorophyll synthesis. The Arabidopsis porB-1 porC-1 mutant displays a severe xantha (highly chlorophyll-deficient) phenotype characterized by smaller prolamellar bodies in etioplasts and decreased thylakoid stacking in chloroplasts. Here we have demonstrated the ability of an ectopic PORA overexpression construct to restore prolamellar body formation in the porB-1 porC-1 double mutant background. In response to illumination, light-dependent chlorophyll production, thylakoid stacking and photomorphogenesis are also restored in PORA-overexpressing porB-1 porC-1 seedlings and adult plants. An Arabidopsis porB-1 porC-1 double mutant can therefore be functionally rescued by the addition of ectopically expressed PORA, which suffices in the absence of either PORB or PORC to direct bulk chlorophyll synthesis and normal plant development.

LHPP, the light-harvesting NADPH:protochlorophyllide (Pchlide) oxidoreductase:Pchlide complex of etiolated plants, is developmentally expressed across the barley leaf gradient

NADPH:protochlorophyllide oxidoreductase is a key enzyme for the light-induced greening of etiolated angiosperm plants. In barley, two POR proteins exist termed PORA and PORB that have previously been proposed to structurally and functionally cooperate in terms of a higher molecular mass light-harvesting complex named LHPP, in the prolamellar body of etioplasts [Nature 397 (1999) 80]. In this study we examined the expression pattern of LHPP during seedling etiolation and de-etiolation under different experimental conditions. Our results show that LHPP is developmentally expressed across the barley leaf gradient. We further provide evidence that LHPP operates both in plants that etiolate completely before being exposed to white light and in plants that etiolate only partially and begin light-harvesting as soon as traces of light become available in the uppermost parts of the soil. As a result of light absorption, in either case LHPP converts Pchlide a to chlorophyllide (Chlide) a and in turn disintegrates. The released Chlide a, as well as Chlide b produced upon LHPP’s light-dependent dissociation, which leads to the activation of the PORA as a Pchlide b-reducing enzyme, then bind to homologs of water-soluble chlorophyll proteins of Brassicaceae. We propose that these proteins transfer Chlide a and Chlide b to the thylakoids, where their esterification with phytol and assembly into the photosynthetic membrane complexes ultimately takes place. Presumably due to the tight coupling of LHPP synthesis and degradation, as well as WSCP formation and photosynthetic membrane assembly, efficient photo-protection is conferred onto the plant.

Fluorescence of the Protein of the Prolamellar Bodies of Etioplasts

In the high-purity membranes of the prolamellar bodies (PLB) of etioplasts, the main protein component of which is protochlorophyllide oxidoreductase (POR), “protein” fluorescence in the 320–350-nm region with a maximum in the excitation spectrum at 278–280 nm has been revealed. The microsurrounding of protein fluorophors in an acidic medium (pH 4.0) is the most hydrophobic. Transfer of energy from the protein to pyrene present in the lipid of the PLB membranes is observed, but fluorescence of protochlorophyllide (Pd) is absent when pyrene is excited via protein. A change in the content of Pd and carotenoids in the membranes of PLB influences the degree of excimerization of the acceptor molecules of pyrene (inverse dependence). The character of interaction of POR with a membrane with allowance for the data on its primary and secondary structures is discussed.

In Situ Conversion of Protochlorophyllideb to Protochlorophyllide a in Barley: EVIDENCE FOR A NOVEL ROLE OF 7-FORMYL REDUCTASE IN THE PROLAMELLAR BODY OF ETIOPLASTS

We recently put forth a model of a protochlorophyllide (Pchlide) light-harvesting complex operative during angiosperm seedling de-etiolation (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80-84). This model, which was based on in vitro reconstitution experiments with zinc analogs of Pchlide a and Pchlide b and the two NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, of barley, predicted a 5-fold excess of Pchlide b, relative to Pchlide a, in the prolamellar body of etioplasts. Recent work (Scheumann, V., Klement, H., Helfrich, M., Oster, U., Schoch, S., and Rüdiger, W. (1999) FEBS Lett. 445, 445-448), however, contradicted this model and reported that Pchlide b would not be present in etiolated plants. Here we demonstrate that Pchlide b is an abundant pigment in barley etioplasts but is rather metabolically unstable. It is rapidly converted to Pchlide a by virtue of 7-formyl reductase activity, an enzyme that had previously been implicated in the chlorophyll (Chl) b to Chl a reaction cycle. Our findings suggest that etiolated plants make use of 7-formyl reductase to fine tune the levels of Pchlide b and Pchlide a and thereby may regulate the steady-state level of light-harvesting POR-Pchlide complex.

Chloroplast structure: from chlorophyll granules to supra-molecular architecture of thylakoid membranes.

This review provides a brief historical account of how microscopical studies of chloroplasts have contributed to our current knowledge of the structural and functional organization of thylakoid membranes. It starts by tracing the origins of the terms plastid, grana, stroma and chloroplasts to light microscopic studies of 19th century German botanists, and then describes how different types of electron microscopical techniques have added to this field. The most notable contributions of thin section electron microscopy include the elucidation of the 3-D organization of thylakoid membranes, the discovery of prolamellar bodies in etioplasts, and the structural changes in thylakoid architecture that accompany the light-dependent transformation of etioplasts into chloroplasts. Attention is then focused on the roles that freeze-fracture and freeze-etch electron microscopy and immuno electron microscopy have played in defining the extent to which the functional complexes of thylakoids are non-randomly distributed between appressed, grana and non-appressed stroma thylakoids. Studies reporting on how this lateral differentiation can be altered experimentally, and how the spatial organization of functional complexes is affected by alterations in the light environment of plants are also included in this discussion. Finally, the review points to the possible uses of electron microscope tomography techniques in future structural studies of thylakoid membranes.

The Extra Loop Distinguishing POR from the Structurally Related Short-chain Alcohol Dehydrogenases Is Dispensable for Pigment Binding but Needed for the Assembly of Light-harvesting POR-Protochlorophyllide Complex

We have recently discovered a protochlorophyllide (Pchlide)-based light-harvesting complex involved in chlorophyll a biosynthesis. This complex consists of the two previously identified NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, their natural substrates (Pchlide b and Pchlide a, respectively), plus NADPH. These are all held together in a stoichiometry of five PORA-Pchlide b-NADPH complexes and one PORB-Pchlide a-NADPH complex in the prolamellar body of etioplasts. The assembly of this novel light-harvesting POR-Pchlide complex (LHPP) requires both the proper interaction of the PORA and PORB with their cognate substrates as well as the oligomerization of the resulting POR-pigment-NADPH ternary complexes into the native, lipid-containing structure of the etioplast. In this study, we demonstrate that the conserved extra sequence that distinguishes PORA and PORB from the structurally related short-chain alcohol dehydrogenases, is dispensable for pigment binding but needed for the assembly of LHPP. As shown by in vitro mutagenesis, deleting this extra sequence gave rise to assembly-incompetent but pigment-containing PORA and PORB polypeptides.

In Vitro Reconstitution of Light-harvesting POR-Protochlorophyllide Complex with Protochlorophyllides a and b

NADPH:protochlorophyllide oxidoreductase (POR; EC ) is a key enzyme for the light-induced greening of angiosperms. In barley, two POR proteins exist, termed PORA and PORB. These have previously been proposed to form higher molecular weight light-harvesting complexes in the prolamellar body of etioplasts (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80-84). Here we report the in vitro reconstitution of such complexes from chemically synthesized protochlorophyllides (Pchlides) a and b and galacto- and sulfolipids. Low temperature (77 K) fluorescence measurements revealed that the reconstituted, lipid-containing complex displayed the same characteristics of photoactive Pchlide 650/657 as the presumed native complex in the prolamellar body. Moreover, Pchlide F650/657 was converted to chlorophyllide (Chlide) 684/690 upon illumination of the reconstituted complex with a 1-ms flash of white light. Identification and quantification of acetone-extractable pigments revealed that only the PORB-bound Pchlide a had been photoactive and was converted to Chlide a, whereas Pchlide b bound to the PORA remained photoinactive. Nondenaturing PAGE of the reconstituted Pchlide a/b-containing complex further demonstrated a size similar to that of the presumed native complex in vivo, suggesting that both complexes may be identical.

A protochlorophyllide-dependent protein import complex of chloroplasts

Chloroplasts synthesize an abundance of different tetrapyrrole compounds. Among them are chlorophyll and its precursor protochlorophyllide, which accumulate in light- and dark-grown plants, respectively. Protochlorophyllide is bound to two NADPH:Pchlide oxidoreductases (POR), termed PORA and PORB, which in angiosperms form higher molecular weight complexes in the prolamellar body of etioplasts. Both POR proteins are nuclear gene products which must be imported from the cytosol.. Import of the precursor of the PORA requires Pchlide and is due to the operation of a second, substrate-dependent protein import site in the plastid envelope. Here we report the isolation of components of the Pchlide-dependent import (PIM) complex and show that it is composed of at least 12 different polypeptides. Among them is a Pchlide a oxygenase which provides Pchlide b as the import substrate of the pPORA, and a tyrosine amino transferase presumably involved in conferring photoprotection onto the PIM complex. Two other constituents were found to be related to previously characterized outer envelope membrane proteins: a 33K protein to TOC33 which is a GTP binding protein, and a 16K protein to a voltage-dependent, amino acid-sective channel protein in the outer plastid envelope.

A protochlorophyllide light-harvesting complex involved in de-etiolation of higher plants

When etiolated angiosperm seedlings break through the soil after germination, they are immediately exposed to sunlight, but at this stage they are unable to perform photosynthesis1. In the absence of chlorophyll a and chlorophyll b, two other porphyrin species cooperate as the basic light-harvesting structure of etiolated plants. Protochlorophyllide a and protochlorophyllide b (ref. 2) form supramolecular complexes with NADPH and two closely related NADPH:protochlorophyllide oxidoreductase (POR) proteins—PORA and PORB (ref. 3)—in the prolamellar body of etioplasts. Here we report that these light-harvesting POR–protochlorophyllide complexes, named LHPP, are essential for the establishment of the photosynthetic apparatus and also confer photoprotection on the plant. They collect sunlight for rapid chlorophyll a biosynthesis and, simultaneously, dissipate excess light energy in the bulk of non-photoreducible protochlorophyllide b. Based on this dual function, it seems that LHPP provides the link between skotomorphogenesis and photosynthesis that is required for efficient de-etiolation.

Structural genes for Mg-chelatase subunits in barley: Xantha-f, -g and -h.

Barley mutants in the loci Xantha-f, Xantha-g and Xantha-h, when fed with 5-aminolevulinate in the dark, accumulate protoporphyrin IX. Mutant alleles at these loci that are completely blocked in protochlorophyllide synthesis are also blocked in development of prolamellar bodies in etioplasts. In contrast to wild type, the xan-f, -g and -h mutants had no detectable Mg-chelatase activity, whereas they all had methyltransferase activity for synthesis of Mg-protoporphyrin monomethyl ester. Antibodies recognising the CH42 protein of Arabidopsis thaliana and the OLIVE (OLI) protein of Antirrhinum majus immunoreacted in wild-type barley with 42 and 150 kDa proteins, respectively. The xan-h mutants lacked the protein reacting with antibodies raised against the CH42 protein. Two xan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to the A. majus olive and the A. thaliana Ch-42 genes were cloned using PCR and screening of cDNA and genomic libraries. Probes for these genes were applied to Northern blots of RNA from the xantha mutants and confirmed the results of the Western analysis. The mutants xan-f27, -f40, -h56 and -h57 are defective in transcript accumulation while -h38 is defective in translation. Southern blot analysis established that h38 has a deletion of part of the gene. Mutants xan-f10 and -f41 produce both transcript and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded that X an-f -h genes encode two subunits of the barley Mg-chelatase and that X an-g is likely to encode a third subunit. The XAN-F protein displays 82% amino acid sequence identity to the OLI protein of Antirrhinum, 66% to the Synechocystis homologue and 34% identity to the Rhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identity to the Arabidopsis CH42 protein, 69% identity to the Euglena CCS protein, 70% identity to the Cryptomonas BchA and Olisthodiscus CssA proteins, as well as 49% identity to the Rhodobacter BchI subunit of Mg-chelatase. Identification of the barley X an-f and X an-h encoded proteins as subunits required for Mg-chelatase activity supports the notion that the Antirrhinum OLI protein and the Arabidopsis Ch42 protein are subunits of Mg-chelatase in these plants. The expression of both thet X an-f and -h genes in wild-type barley is light induced in leaves of greening seedlings, and in green tissue the genes are under the control of a circadian clock.

Prolamellar-body structure, composition of molecular species and amount of galactolipids in etiolated, greening and reetiolated primary leaves of oat, wheat and rye

The greening and reetiolation process of etiolated leaves of oat, wheat and rye, possessing different types of prolamellar bodies (PLBs), was observed by electron microscopy. Oat is known to possess unusual crystalline PLBs (so-called 'narrow type'). Rye and what, which normally show PLBs with more loosely packed tubules ('wide type') during etiolation, exhibited PLBs of the narrow type after illumination and subsequent reincubation in the dark (=reetiolation). Thus the reetiolated PLBs of wheat and rye did not differ from etiolated or reetiolated oat PLBs. In parallel with the microscopic analysis, intact leaves of all developmental stages were analysed for their galactolipid content and composition of molecular species using a newly developed high-performance liquid chromatography procedure. When oat, wheat and rye were compared, differences in the molecular species and the molar ratio of the two galactolipids monogalactosyldiacylglyceride (MGDG) and digalactosyldiacylglyceride (DGDG) were found. However, no parameter showed a correlation with PLB construction, disintegration or reconstruction. The results presented in this paper are not consistent with the hypothesis that the molar ratio of MGDG/DGDG is responsible for the tubular structure of prolamellar bodies in etioplasts.

Prolamellar body and saponins: Avenacosides are not constituents of Avena etioplasts

Etiolated Avena leaf cells were homogenized, then fractionated into four fractions in the presence of salts by differential centrifugation, and intact etioplasts were prepared by Percoll or sucrose density gradient centrifugation. Using thin layer chromatography steroidal saponins, avenacoside A and B were found in the leaf cells and heavy cell fractions which were rich in other cell structures besides intact etioplasts, but not detected in the purified etioplasts. We concluded that saponins are not constituents of the prolamellar bodies in etioplasts.

Physiology and plastid fine structure of deetiolating Lemna minor

During the 12-h lag period in chlorophyll accumulation after the onset of white-light illumination of Lemna minor etiolated for 35 days, a rapid increase in visible fronds per culture occurred. This new frond production then assumed a log-linear rate of increase, and total protein per unit fresh weight came to parallel the rate of increase in fresh weight per plant. The ribosomal RNA content of 45-day-etiolated plants was deficient in 23S and 16S species compared with green plants. The prolamellar bodies of etioplasts were either tightly or loosely paracrystalline within the same cell; they were without extended perforate lamellae, which developed during far-red-light illumination even while prolamellar bodies persisted. The development of chloroplasts in deetiolating L. minor was typical of other higher plants. The developmental sequence in green Lemna included proplastid to deeply stacked granal chloroplast within several millimetres. Plastid profiles suggestive of division configurations occurred only in primordial cells of green and etiolated plants. The relatively small numbers of plastids in any given stage of differentiation may account for the sensitivity of plastid development to inhibitors of protein and nucleic acid synthesis.