Localization of Galactolipid Biosynthesis in Etioplasts Isolated from Dark-Grown Wheat (Triticum aestivum L.)

Etioplasts were isolated from leaves of dark-grown wheat (Triticum aestivum L. var Starke II). Galactolipid biosynthesis was assayed in an envelope-rich fraction and in the fraction containing the rest of the etioplast membranes by measuring incorporation of ¹⁴C from uridine-diphospho[¹⁴C]galactose into monogalactosyl diacylglycerol and digalactosyl diacylglycerol. More than half of the galactolipid biosynthetic capability was found in the fraction of inner etioplast membranes. This fraction was subfractioned into fractions enriched in prolamellar bodies and membrane vesicles (prothylakoids), respectively. All membrane fractions obtained from etioplasts were able to carry out galactolipid biosynthesis, although the activity was very low in prolamellar body-enriched fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed markedly different polypeptide patterns between the different fractions. It is concluded that the capability of galactolipid biosynthesis of etioplasts probably is not restricted to the envelope, but is also present in the inner membranes of this plastid.     

Stimulation of Mitochondrial Calcium Uptake by Light during Growth of Corn Shoots

Ca²⁺ uptake in mitochondrial fractions, isolated on Percoll discontinuous density gradients, from light- and dark-grown corn (Zea mays L. var W64A × W182E) shoots was characterized by dual wavelength spectroscopy and the Ca²⁺-sensitive dye murexide. In light-grown seedlings, the rate of mitochondrial Ca²⁺ uptake was about 40 nanomoles per minute per milligram of mitochondrial protein. A portion of the Ca²⁺ uptake required an exogenous supply of ATP (65%) while the remaining 35% was the respiratory substrate-dependent reaction. Ruthenium red (2 micromolar) completely inhibited both ATP- and substrate-dependent reactions. There was no detectable Ca²⁺ efflux from the mitochondria with the inhibitor. When the mitochondrial fraction was prepared from the dark-grown shoots, the rate of uptake, in particular the ATP-dependent reaction, was greatly reduced. The dark treatment caused a reduction in mitochondrial Ca content which is largely due to the reduction of Ca associated with the mitochondrial membrane rather than to a reduction of Ca in the soluble matrix.     

Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

Behavior of the 36,000-dalton Protein in the Internal Membranes of Squash Etioplasts during Greening

The 36,000-dalton protein was found to be a major componentin the internal membrane of etioplasts of dark-grown squashcotyledons by slab SDS-polyacrylamide gel electrophoresis. Itwas also a major membrane protein of the etioplast in otheretiolated plants such as oat and maize. When the etioplastswere transformed into (etio)chloroplasts under continued illumination,depletion of the 36,000-dalton protein began without a lag timefollowed by their disappearance, while formation of apoproteinsof the light-harvesting chlorophyll protein complex, 26,000-and 22,400-dalton proteins, was noted. A 55,000-dalton proteinwas also present and its content did not change during greening.O'Farrell's two-dimensional electrophoresis revealed that the36,000-dalton protein consisted of at least four alkaline proteinsof the same molecular weight but with different isoelectricpoints of 9.1, 8.8, 8.5 and 8.2. They were found to be verysimilar protein by partial proteolysis using staphylococcalprotease. The two-dimensional electrophoresis pattern of the36,000-dalton protein was altered after 1 hr illumination. Thesebehaviors of the 36,000-dalton protein during the greening processwere accompanied by transformation of the crystalline prolamellarbody in the etioplast. (Received September 16, 1981; Accepted March 15, 1982)     

Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles.

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.     

Proteome analysis of the rice etioplast: metabolic and regulatory networks and novel protein functions

We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.     

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Effect of light on ribonucleic Acid metabolism in greening maize leaves

The effect of illumination on the incorporation of labeled precursors into RNA of dark-grown maize (Zea mays) leaves was studied using either ³²P-phosphate or double labeling with ¹⁴C- and ³H-uridine. In the dark, label was preferentially incorporated into etioplast ribosomal RNAs. Incorporation into this fraction and into lower molecular weight fractions was strongly and preferentially stimulated by light during the first 2 hours of illumination. The effect persisted after illumination was terminated. The possibility that light-induced alterations in plastid ribosomal RNA metabolism may not be required for chlorophyll accumulation in maize is discussed.     

The significance of promitochondrial structures in rat liver for mitochondrial biogenesis

1. The heavy, light and fluffy mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their protein synthesizing ability in vitro, their nucleic acid content, buoyant density of their DNA and ultrastructure. 2. The light mitochondrial fraction synthesized proteins in vitro at a rate 4-5 times as high as heavy and fluffy mitochondria. The incorporation ability of this fraction was also maximally affected by the thyroid status of the animal. The radioactivity in leucyl-tRNA of the light mitochondrial fraction was about 3-4 times as high as that of the other two fractions. 3. The heavy, light and fluffy mitochondrial fractions contained small but consistent amounts of RNA and DNA. Although the DNA content was the same in all mitochondria fractions, the light mitochondria contained relatively more RNA. The buoyant density of DNA from all the fractions was 1.701g/cm(3). 4. Electron microscopy revealed that the heavy mitochondria have a typical mitochondrial architecture, with densely packed cristae and a well developed double membrane. Light mitochondria were also surrounded by double membranes, but were smaller in size and contained less cristae. The fluffy fraction consisted of a mixture of well formed mitochondria and those in the process of degradation. 5. The significance of these findings in relation to mammalian mitochondrial genesis is discussed.     

Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley

In barley (Hordeum vulgare L.) root cells, activity for oxidizing protoporphyrinogen to protoporphyrin (protoporphyrinogen oxidase), a step in chlorophyll and heme synthesis, was found both in the crude mitochondrial fraction and in a plasma membrane enriched fraction separated by a sucrose gradient technique utilized for preparing plasma membranes. The specific activity (expressed as nanomoles of protoporphyrin formed per hour per milligram protein) in the mitochondrial fraction was 8 and in the plasma membrane enriched fraction was 4 to 6. The plasma membrane enriched fraction exhibited minimal cytochrome oxidase activity and no carotenoid content, indicating little contamination with mitochondrial or plastid membranes. Etioplasts from etiolated barley leaves exhibited a protoporphyrinogen oxidase specific activity of 7 to 12. Protoporphyrinogen oxidase activity in the barley root mitochondrial fraction and etioplast extracts was more than 90% inhibited by assay in the presence of the diphenyl ether herbicide acifluorfen methyl, but the activity in the plasma membrane enriched fraction exhibited much less inhibition by this herbicide (12 to 38% inhibition) under the same assay conditions. Acifluorfen-methyl inhibition of the organellar (mitochondrial or plastid) enzyme was maximal upon preincubation of the enzyme with 4 mm dithiothreitol, although a lesser degree of inhibition was noted if the organellar enzyme was preincubated in the presence of other reductants such as glutathione or ascorbate. Acifluorfen-methyl caused only 20% inhibition if the enzyme was preincubated in buffer without reductants. Incubation of barley etioplast extracts with the earlier tetrapyrrole precursor coproporphyrinogen and acifluorfen-methyl resulted in the accumulation of protoporphyrinogen, which could be converted to protoporphyrin even in the presence of the herbicide by the addition of the plasma membrane enriched fraction from barley roots. These findings have implications for the toxicity of diphenyl ether herbicides, whose light induced tissue damage is apparently caused by accumulation of the photoreactive porphyrin intermediate, protoporphyrin, when the organellar protoporphyrinogen oxidase enzyme is inhibited by herbicides. Our results suggest that the protoporphyrinogen that accumulates as a result of herbicide inhibition of the organellar enzyme can be oxidized to protoporphyrin by a protoporphyrinogen oxidizing activity that is located at sites such as the plasma membrane, which is much less sensitive to inhibition by diphenylether herbicides.     

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tangerås, A., Flatmark, T., Bäckström, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162–175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04±0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37°C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.     

Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations

Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm³ and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm³. During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm³. However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm³ and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm³ was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.     

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tanger?s, A., Flatmark, T., B?ckstr?m, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162-175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04 +/- 0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37 degrees C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.     

A comparison of prolamellar bodies from wheat, Scots pine and Jeffrey pine. Pigment spectra and properties of protochlorophyllide oxidoreductase

Inner etioplast membrane fractions were isolated from wheat ( Triticum aestivum L. cv. Starkell), Scots pine ( Pinus sylvestris L.) and Jeffrey pine ( Pinus jeffreyi Murr), in order to investigate whether cotyledons of dark-grown conifers have protochlorophyllide associated to protochlorophyllide oxidoreductase (EC 1.6.99.–) in the pro-lamellar body in the same way as angiosperms. Protochlorophyllide was found to be present in dark-grown seedlings of Scots pine and Jeffrey pine to the same extent as in dark-grown wheat, 10–15.8 nmol (g fresh weight)⁻¹. Fluorescence emission spectra at 77 K showed accumulation of protochlorophyllide with emission maximum at 657 nm in the prolamellar body fractions of the three species studied. Also the light- and NADPH-dependent activity of protochlorophyllide oxidoreductase was consistently localized in the prolamellar body fractions. The three prolamellar body fractions were dominated by the same polypeptide. Its molecular weight was estimated to be 38 000 by sodium dodecylsulphate polyacrylamide gel electrophoresis.     

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins’ largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.     

Frataxin-mediated iron delivery to ferrochelatase in the final step of heme biosynthesis

Human ferrochelatase, a mitochondrial membrane-associated protein, catalyzes the terminal step of heme biosynthesis by insertion of ferrous iron into protoporphyrin IX. The recently solved x-ray structure of human ferrochelatase identifies a potential binding site for an iron donor protein on the matrix side of the homodimer. Herein we demonstrate Hs holofrataxin to be a high affinity iron binding partner for Hs ferrochelatase that is capable of both delivering iron to ferrochelatase and mediating the terminal step in mitochondrial heme biosynthesis. A general regulatory mechanism for mitochondrial iron metabolism is described that defines frataxin involvement in both heme and iron-sulfur cluster biosyntheses. In essence, the distinct binding affinities of holofrataxin to the target proteins, ferrochelatase (heme synthesis) and ISU (iron-sulfur cluster synthesis), allows discrimination between the two major iron-dependent pathways and facilitates targeted heme biosynthesis following down-regulation of frataxin.     

Hydroxymethylglutaryl coenzyme A reductase activity in hamster adrenal mitochondria purified on a sucrose gradient.

Hamster adrenal homogenates were fractionated by differential centrifugation to obtain crude mitochondrial and microsomal pellets. The mitochondria were further purified on a linear sucrose density gradient. The crude mitochondrial fraction was separated into three bands on the gradient. One of the bands (band 3, D₂₀²⁰ = 1.165) contained all the measurable cytochrome C oxidase activity. Band 3 also contained the highest specific activity of HMG-CoA reductase corresponding to a 1.9 fold enrichment compared to the crude mitochondrial pellet. The evidence presented supports the possibility that a part of the HMG-CoA reductase activity in hamster adrenals is associated with mitochondria.     

Characterization of prolamellar bodies, from dark-grown seedlings of Scots pine, containing light- and NADPH-dependent protochlorophyllide oxidoreductase

Cotyledons of conifers have a light-independent pathway for chlorophyll biosynthesis. To investigate whether the prolamellar body of Scots pine ( Pinus sylveslris L.) is similar to the better known prolamellar body of wheat, etioplast membrane fractions were isolated from cotyledons of dark-grown Scots pine. Dark-grown cotyledons contained both chlorophyll and protochlorophyllide, 158 and 10 nmol (g fresh weight)'respectively, and had a chlorophyll a to b ratio of 4.2. The content of glyco- and phospholipids was 7.1 μmol (g fresh weight)¹. About 40 mol % of these lipids were the specific plastid lipids – monogalactosyl diacylglycerol. digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol in the relative amounts 50, 35 and 7 mol %. The mol ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was 1.7. Low temperature fluorescence emission spectra of intact cotyledons and homogenate showed maxima at 633, 657, 686, 696 nm and a broad peak at 725–735 nm. The maxima at 633 and 657 nm represented different forms of protochlorophyllide and the other emission maxima represented chlorophyll protein complexes. The 657 nm form of protochlorophyllide was phototransformable both in vivo and in the isolated membranes. The phototransformable protochlorophyllide was substantially enriched in the prolamellar body fraction.
The specific activity of light dependent protochlorophyllide oxidoreductase in the prolamellar body fraction was found to be 2 nmol chlorophyllide formed [(mg protein)⁻¹ min⁻¹]. The molecular weight of the enzyme polypeptide was determined as 38 000 dalton with sodium dodecylsulphate-polyacrylamide gel electrophoresis.     

Immunochemical studies of ferrochelatase protein: characterization of the normal and mutant protein in bovine and human protoporphyria.

Protoporphyria is a hereditary disorder characterized by a marked decrease in the activity of ferrochelatase, the terminal enzyme in the heme biosynthetic pathway. We have prepared specific polyvalent antibodies against bovine ferrochelatase in rabbits. The specificity of the antibody preparation against ferrochelatase was demonstrated by western blot analysis and immunoprecipitation of ferrochelatase activity. The antibody also cross-reacted weakly with ferrochelatase from human mitochondria. To quantify immunoreactive ferrochelatase in tissue samples, a kinetic-based enzyme-linked immunosorbent assay (k-ELISA) was developed. Ferrochelatase activity and the level of immunoreactive protein were measured in hepatic mitochondria isolated from six normal and nine protoporphyric (homozygous) cattle. Ferrochelatase activity was less than 10% of normal in mitochondria from protoporphyric animals; the amount of immunoreactive material was equivalent to that from normal animals. Similar studies were performed with samples from three normal and two protoporphyric (heterozygous) humans. Ferrochelatase activity was decreased in protoporphyric samples (about 17% of normal, but there was no concomitant decrease in immunoreactive material. These data demonstrate that a normal amount of ferrochelatase protein is present and suggest that bovine and human protoporphyria result from point mutations in the gene encoding ferrochelatase.