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PubMed1967

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris. I. Localization of Activity in the Chloroplasts of a Chloroplast Containing Fraction from Developing Leaves.

The incorporation of uniformly labeled leucine-(14)C into protein by a chloroplast containing fraction from developing primary leaves of bean is reported. Chloroplasts, obtained from week old plants grown in darkness, and then illuminated with white light for 12 hours, were shown to be the principal sites of incorporating activity. Incorporation may continue for 2 hours. Rates of up to 50 mumumole leucine incorporated per mg protein per hour are observed when a 1 hour assay period is used. Incorporation is only partially sensitive to ribonuclease.

Journal Article↗
PubMed1972

Studies on Chloroplast Development and Replication in Euglena: III. A Study of the Site of Synthesis of Alkaline Deoxyribonuclease Induced during Chloroplast Development in Euglena gracilis.

During chloroplast development in Euglena, the activity of a specific DNase, Euglena alkaline DNase, increases in a manner similar to that of chlorophyll synthesis, but without the lag customarily associated with the early hours of chlorophyll synthesis. The increase in Euglena alkaline DNase activity is not inhibited by chloramphenicol or by streptomycin, but is inhibited by cycloheximide. Euglena alkaline DNase activity is present in a group of aplastidic substrains which contain carotenoids. These results are interpreted to mean that this chloroplast-related DNase is synthesized in the cytoplasm, and that the genetic information for this enzyme is probably nuclear.It is also shown that different bleached substrains exhibit substantial variation, both in total carotenoids and in Euglena alkaline DNase activity. These results are discussed in terms of the possibility that a cytoplasmic photoreceptor system is influencing the light-induced increase in Euglena alkaline DNase activity.

Journal Article↗
PubMed1982

Chloroplast DNA levels and the control of chloroplast division in light-grown wheat leaves.

Plastids at different stages of development were isolated from light-grown wheat (Triticum aestivum, var. Maris Dove) seedling leaves, and the average chloroplast DNA (cpDNA) per plastid at each developmental stage was measured directly. In the earliest stages of development, the number of plastids per cell and the amount of cpDNA per cell increased with cell age, but cpDNA per plastid remained constant at between 800 and 1,000 genome copies per plastid. After this phase, plastids per cell continued to increase, but cpDNA per plastid decreased. Subsequently, both plastids per cell and cpDNA per plastid remained constant as cell age increased, the final DNA content being approximately 300 genome copies per plastid. These results are related to previous reports of cpDNA changes during the development of dicotyledonous plants, and to theories about the regulation of chloroplast numbers per cell.

Journal Article↗
PubMed1988

Ion Homeostasis in Chloroplasts under Salinity and Mineral Deficiency : I. Solute Concentrations in Leaves and Chloroplasts from Spinach Plants under NaCl or NaNO(3) Salinity.

Spinach (Spinacia oleracea var "Yates") plants in hydroponic culture were exposed to stepwise increased concentrations of NaCl or NaNO(3) up to a final concentration of 300 millimoles per liter, at constant Ca(2+)-concentration. Leaf cell sap and extracts from aqueously isolated spinach chloroplasts were analyzed for mineral cations, anions, amino acids, sugars, and quarternary ammonium compounds. Total osmolality of leaf sap and photosynthetic capacity of leaves were also measured. For comparison, leaf sap from salt-treated pea plants was also analyzed. Spinach plants under NaCl or NaNO(3) salinity took up large amounts of sodium (up to 400 millimoles per liter); nitrate as the accompanying anion was taken up less (up to 90 millimoles per liter) than chloride (up to 450 millimoles per liter). Under chloride salinity, nitrate content in leaves decreased drastically, but total amino acid concentrations remained constant. This response was much more pronounced (and occurred at lower salt concentrations) in leaves from the glycophyte (pea, Pisum sativum var "Kleine Rheinländerin") than from moderately salt-tolerant spinach. In spinach, sodium chloride or nitrate taken up into leaves was largely sequestered in the vacuoles; both salts induced synthesis of quarternary ammonium compounds, which were accumulated mainly in chloroplasts (and cytosol). This prevented impairment of metabolism, as indicated by an unchanged photosynthetic capacity of leaves.

Journal Article↗
PubMed1993

Posttranscriptional accumulation of chloroplast tufA (elongation factor gene) mRNA during chloroplast development in Chlamydomonas reinhardtii.

Light induces chloroplast (Cp) differentiation in dark-grown y-1 strains of Chlamydomonas reinhardtii. Slot blot analysis was used to quantitate tufA, psbA, psbK, rbcL, and 16S rRNA transcript accumulation and transcription during Cp differentiation. When etiolated cc-125 y-1 cells were illuminated for 5 h, a 1710 bp tufA mRNA accumulated up to 5-fold while the psbA, rbcL, and 16S rRNA transcripts accumulated less than 1.5-fold. The tufA gene encodes translational elongation factor EF-Tu. The light-induced accumulation of tufA mRNA did not occur in cc-1931, a strain that does not become etiolated in darkness. Pulse labelling was used to measure the transcription of Cp transcripts during tufA mRNA accumulation, and no detectable change in tufA transcription was observed. These results imply that the half life of the tufA transcript increases during the greening process.

Amino Acid Sequence↗
PubMed1991

Transfer of defined numbers of chloroplasts into albino protoplasts using an improved subprotoplast/protoplast microfusion procedure: transfer of only two chloroplasts leads to variegated progeny.

A procedure is described by which it is possible to perform controlled microfusion of microscopically selected protoplast fusion partners with high efficiencies. The procedure is applied to fusion of Nicotiana tabacum (line 92V37. N. undulata cytoplasm) plastid albino protoplasts as a recipient and spontaneously formed subprotoplasts of green N. tabacum (line SR1) as donor. Products of individual electrofusion events are cloned via single cell nurse culture and the derived cell lines are analysed for the occurrence of variegated or green regenerating shoots, which are indicative of the establishment of the transferred organelles in the cell progeny. The plastid population in green regenerants recovered after the transfer of only two chloroplasts was demonstrated to have originated from the donor subprotoplast organelles by restriction analysis of total DNA using a plastome-specific probe.

Blotting, Southern↗
PubMed2001

Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach. Cyanobacterial genes for chloroplast and cytosolic isoenzymes encoded in eukaryotic chromosomes.

Previous attempts to purify chloroplast 6-phosphogluconate dehydrogenase (cp6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been unsuccessful due to rapid activity loss. An efficient purification protocol was developed and the enzyme from spinach leaves was purified 1000-fold to apparent homogeneity with a specific activity of 60 U.mg-1. The enzyme is a homodimer with subunits of 50 kDa. Antibodies raised against the purified cp6PGDH detected a 53-kDa protein from a crude extract, indicating alterations during purification. Purified cp6PGDH was microsequenced and the corresponding spinach cDNA was cloned using PCR techniques and degenerate primers. The cDNA for cytosolic 6PGDH from spinach was cloned for comparison. Phylogenetic analysis in the context of available homologues from eukaryotes and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences are more similar to their homologues from gamma-proteobacteria, whereas plant 6PGDH is more similar to its cyanobacterial homologues. The ancestral gene for higher plant 6PGDH was acquired from the antecedent of plastids through endosymbiosis and gene transfer to the nucleus. A subsequent gene duplication gave rise to higher plant cytosolic 6PGDH, which assumed the function of its pre-existing cytosolic homologue through endosymbiotic gene replacement. The protein phylogeny of both 6PGDH and of the first enzyme of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase, indicate a surprisingly close relationship between the plant and Trypanosoma brucei lineages, suggesting that T. brucei (a relative of Euglena gracilis) may be secondarily nonphotosynthetic.

Amino Acid Sequence↗
PubMed2006

Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling.

Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process. The corresponding product, magnesium protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. To get more information on the chlorophyll biosynthesis pathway and on magnesium protoporphyrin IX derivative functions, we have identified an magnesium protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from magnesium protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cytochrome b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that magnesium protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of magnesium protoporphyrin IX methyl ester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the magnesium chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step.

Arabidopsis↗
PubMed1976

Chloroplast membranes of the green alga Acetabularia mediterranea. II. Topography of the chloroplast membrane.

The localization of the chlorophyll-protein complexes inside the thylakoid membrane of Acetabularia mediterranea was determined by fractionating the chloroplast membrane with EDTA and Triton X-100, by using pronase treatment, and by labeling the surface-exposed proteins with 125I. The effects of the various treatments were established by electrophoresis of the solubilized membrane fractions and electron microscopy. After EDTA and pronase treatment, the membrane structure was still intact. Only the two chlorophyll-protein complexes of 67,000 and 152,000 daltons and an additional polypeptides were found in the membrane before the EDTA and pronase treatment. The 125,000 dalton complex seems to be buried inside the lipid layer. The 23,000 dalton subunit of the 67,000 dalton complex is largely exposed to the surface of the EDTA-insoluble membrane and only the chlorophyll-binding subunit of 21,500 daltons is buried inside the lipid layer.

Acetabularia↗
PubMed1990

Reconstitution of photophosphorylation in EDTA-treated thylakoids by added chloroplast coupling factor 1 (ATPase) and chloroplast coupling factor 1 lacking the delta subunit. Structural or functional?

Upon EDTA treatment thylakoids lose the chloroplast coupling factor 1 (CF1) part of their ATP synthase, CF0CF1, this exposes the proton channel, CF0. The previously established ability of the CF1 subunit delta to block the proton leak through CF0 prompted us to study (a) the ability of complete CF1 and, for comparison, CF1 lacking the delta subunit to block proton leakage and thereby to reconstitute structurally some photophosphorylation activity of the remaining CF0CF1 molecules and (b) their ability to form functional enzymes (functional reconstitution). In order to discriminate between activities caused by added CF1 or CF1(-delta) and remaining CF0CF1, the former were inhibited by chemical modification of subunit beta by N,N'-dicyclohexyl carbodiimide (DCCD) and the latter by tentoxin. We found that added CF1 acted both structurally and functionally while added DCCD-treated CF1 (DCCD-CF1) acted only structurally. In contrast to previous observations, CF1(-delta) and DCCD-CF1(-delta) also acted structurally although the reduction of proton leakage was smaller than with DCCD-CF1. Hence there was no functional reconstitution without subunit delta present. Previous studies indicated that only a small fraction of exposed CF0 is highly conducting and that this small fraction is distinguished by its high affinity for added CF1. The results of this study point rather to a wider distribution of CF0 conductance states and binding affinities.

Cell-Free System↗
PubMed2002

Post-illumination reduction of the plastoquinone pool in chloroplast transformants in which chloroplastic NAD(P)H dehydrogenase was inactivated.

We reported previously that an ndhB gene disruptant, delta ndhB, had the same phenotype as wild-type tobacco plants under normal growth conditions. Two other groups have reported conflicting phenotypes with each other for ndhCKJ operon disruptants. Here, we generated two transformants in which the ndhCKJ operon was disrupted, and found that new transformants had the same phenotype as delta ndhB. After illumination with visible light, all ndh disruptants had higher levels of steady-state fluorescence than wild-type controls when measured under weak light, suggesting that reduction of the plastoquinone pool in ndh disruptants was greater than that in wild-type controls. The weak light itself could not reduce the plastoquinone much, so the reduction in the plastoquinone in the mutant was due to electron donation from stromal reductants generated during illumination with the strong light. These results supported the hypothesis that NAD(P)H dehydrogenase prevents overreduction in chloroplasts and suggested that chlororespiratory oxidase did not function under low light or in the dark.

Chlorophyll↗
PubMedDate not supplied

[Isolation of chloroplasts and chloroplast DNA from pea leaves].

A method of isolating DNA from pea chloroplasts (ch-DNA) in CsCl density gradient is described. DNA preparations are free of 5-methylcytosine and have a melting temperature of 86.5 degrees. Denatured DNA molecules completely reassociate for 3 hours at 60 degrees C. It is concluded that the preparations obtained are pure ch-DNA.

Centrifugation, Density Gradient↗
PubMed1975

Investigation of electron transport in the chloroplasts and their fragments by the ESR method. II. Light-induced interaction of water-soluble nitroxide radical with chloroplasts and chlorophyll containing protein-lipid micelles.

A light-induced reduction of the water-soluble nitroxide radical by chlorophyll in lipid and protein--lipid micelles was demonstrated. In contrast to model systems, in whole chloroplasts the NR is photoreduced by the electrons of the noncyclic electron transport chain. The initiation of cyclic electron transport in light particles, containing only photosystem I, does not lead to photoreduction of NR. When exogenous protein -- human serum albumin -- is added to the light particles, the nitroxide radicals are intensively reduced. The specific role of protein in electron transport from P700 to the exogenous acceptor is discussed.

Binding Sites↗
PubMed1977

Chloroplast biogenesis. Net synthesis of protochlorophyllide from magnesium-protoporphyrin monoester by developing chloroplasts.

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.

Chlorophyll↗
PubMed1977

Chloroplast biogenesis. Net synthesis of protochlorophyllide from protoporphyrin IX by developing chloroplasts.

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.

Chlorophyll↗
PubMed1984

Chloroplast tRNA gene contains a long intron in the D stem: Nucleotide sequences of tobacco chloroplast genes for tRNA (UCC) and tRNA (UCU).

The nucleotide sequences of tobacco chloroplast genes for tRNA(Gly) (UCC) and tRNA(Arg) (UCU) have been determined. The tRNA(Gly) gene has a 691-base-pair intron located in the D stem while the tRNA(Arg) gene does not have any intron. The tRNA(Gly) and tRNA(Arg) genes are encoded on the same strand and separated by a 169-base-pair spacer. The tRNA(Gly) gene is transcribed as a 900-base precursor RNA molecule. The tRNA(Gly) and tRNA(Arg) deduced from the DNA sequences show 84% and 55% sequence homologies with Escherichia coli tRNA(Gly) (UCC) and phage T4 tRNA(Arg) (UCU), respectively.

Journal Article↗
PubMed1995

Differential Transcription of Pea Chloroplast Genes during Light-Induced Leaf Development (Continuous Far-Red Light Activates Chloroplast Transcription).

Plastid gene expression was analyzed in pea (Pisum sativum L.) plants grown in darkness, continuous far-red light, and white light. Responses induced by continuous far-red light were most likely mediated by PHYA. Plastid transcription activity was low in dark-grown plants. In contrast, plastids of plants grown in white or far-red light showed a 10-fold increase in transcription activity between 4 and 6 d postimbibition (dpi) and a decrease between 6 and 9 dpi. Plastid RNAs accumulated in illuminated plants from 5 to 7 dpi. In far-red-light-illuminated plants, plastid mRNA levels remained elevated until 14 dpi. In white-light-grown plants, most plastid RNAs decreased in abundance after 7 dpi to very low levels by 14 dpi. This indicates that white light induces a general decrease in plastid RNA stability compared to far-red-light-illuminated seedlings. PsbA mRNA accumulated in older, dark-grown, far-red, and white-light-illuminated seedlings, consistent with this RNA having high stability. Transcription of genes encoding the plastid's transcription and translation apparatus increased relative to rbcL and other genes encoding proteins of the photosynthetic apparatus from 4 to 5 dpi and then declined 10-fold from 5 to 9 dpi. These data document dynamic modulation of plastid gene transcription and mRNA stability during light-induced chloroplast development in pea.

Journal Article↗