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PubMed1969

Studies on chloroplast development and replication in Euglena. I. Vitamin B12 and chloroplast replication.

When Euglena gracilis is grown under vitamin B(12) deficiency conditions, the amount of protein and of chlorophyll per cell increase with decrease of B(12) in the medium and consequently in the cell. The increase in cell protein is proportional to and precedes an increase in the number of chloroplasts per cell. This replication of the chloroplasts under deficiency conditions is not accompanied by nuclear or cell division. It is concluded that chloroplast replication in Euglena gracilis is independent of nuclear and cellular replication, at least under B(12) deficiency conditions. We established a graph of the growth of Euglena under different concentrations of vitamin B(12) added to the growth medium, which permitted us to calculate that at least 22,000 molecules of vitamin B(12) per cell are required to give normal growth.

Cell Division↗
PubMed1997

Both RNA editing and RNA cleavage are required for translation of tobacco chloroplast ndhD mRNA: a possible regulatory mechanism for the expression of a chloroplast operon consisting of functionally unrelated genes.

Tobacco chloroplast genes encoding a photosystem I component (psaC) and a NADH dehydrogenase subunit (ndhD) are transcribed as a dicistronic pre-mRNA which is then cleaved into short mRNAs. An RNA protection assay revealed that the cleavage occurs at multiple sites in the intercistronic region. There are two possible initiation codons in the tobacco ndhD mRNA: the upstream AUG and the AUG created by RNA editing from the in-frame ACG located 25 nt downstream. Using the chloroplast in vitro translation system, we found that translation begins only from the edited AUG. The extent of ACG to AUG editing is partial and depends on developmental and environmental conditions. In addition, the in vitro assay showed that the psaC/ndhD dicistronic mRNA is not functional and that the intercistronic cleavage is a prerequisite for both ndhD and psaC translation. Using a series of mutant mRNAs, we showed that an intramolecular interaction between an 8 nt sequence in the psaC coding region and its complementary 8 nt sequence in the 5' ndhD UTR is the negative element for translation of the dicistronic mRNA. A possible mechanism in which the differential expression of the chloroplast operon consists of functionally unrelated genes is discussed.

Chloroplasts↗
PubMed1998

Genetic analysis of chloroplast c-type cytochrome assembly in Chlamydomonas reinhardtii: One chloroplast locus and at least four nuclear loci are required for heme attachment.

Chloroplasts contain up to two c-type cytochromes, membrane-anchored cytochrome f and soluble cytochrome c6. To elucidate the post-translational events required for their assembly, acetate-requiring mutants of Chlamydomonas reinhardtii that have combined deficiencies in both plastid-encoded cytochrome f and nucleus-encoded cytochrome c6 have been identified and analyzed. For strains ct34 and ct59, where the phenotype displays uniparental inheritance, the mutations were localized to the chloroplast ccsA gene, which was shown previously to be required for heme attachment to chloroplast apocytochromes. The mutations in another eight strains were localized to the nuclear genome. Complementation tests of these strains plus three previously identified strains of the same phenotype (ac206, F18, and F2D8) indicate that the 11 ccs strains define four nuclear loci, CCS1-CCS4. We conclude that the products of the CCS1-CCS4 loci are not required for translocation or processing of the preproteins but, like CcsA, they are required for the heme attachment step during assembly of both holocytochrome f and holocytochrome c6. The ccsA gene is transcribed in each of the nuclear mutants, but its protein product is absent in ccs1 mutants, and it appears to be degradation susceptible in ccs3 and ccs4 strains. We suggest that Ccsl may be associated with CcsA in a multisubunit "holocytochrome c assembly complex," and we hypothesize that the products of the other CCS loci may correspond to other subunits.

Animals↗
PubMed2004

Bcl-2 family members localize to tobacco chloroplasts and inhibit programmed cell death induced by chloroplast-targeted herbicides.

In mammalian cells, apoptosis is often mediated via organelles. While apoptotic-like cell death occurs in plants, the mechanistic details are unresolved. Transgenic tobacco plants have been generated that harbour selected animal anti-apoptotic genes. Subcellular fractionation followed by western blot analysis indicated that chloroplasts serve as a location for these animal anti-apoptotic proteins in addition to the established mitochondrial location. To explore the functional significance of this observation, tobacco plants were treated with three chloroplast-directed herbicides. Wild-type plants died and exhibited features associated with apoptosis. Transgenic plants survived and did not show any apoptotic-like characteristics. Moreover, the herbicide-induced apoptotic-like cell death was light requiring. It was concluded that chloroplasts may be involved in mediating certain types of plant programmed cell death.

Animals↗
PubMed1991

Fine structural features of the chloroplast genome: comparison of the sequenced chloroplast genomes.

The entire nucleotide sequences of the rice, tobacco and liverwort chloroplast genomes have been determined. We compared all the chloroplast genes, open reading frames and spacer regions in the plastid genomes of these three species in order to elucidate general structural features of the chloroplast genome. Analyses of homology, GC content and codon usage of the genes enabled us to classify them into two groups: photosynthesis genes and genetic system genes. Based on comparisons of homology, GC content and codon usage, unidentified ORFs can also be assigned to each of these groups such that it is possible to speculate about the functions of products which may be produced by these ORFs. The spacer regions and intron sequences were compared and found to have no obvious homology between rice and liverwort or between tobacco and liverwort.

Base Composition↗
PubMed1995

The expression of nuclear genes encoding plastid ribosomal proteins precedes the expression of chloroplast genes during early phases of chloroplast development.

The development of different plant organs (root, hypocotyl, and cotyledons) during seed germination is connected with the transformation of proplastids, which are found in embryonic and meristematic tissues, into amyloplasts in root tissues and into chloroplasts in cotyledons. We have analyzed the expression of nuclear and plastid genes coding for the plastid translational apparatus during the first 7 d of Spinacia oleracea development. Results show that the nuclear genes (rps1, rps22, rpI21, and rpI40) are expressed from the 1st d of seed imbibition and precede transcription of the chloroplast-encoded genes (photosynthetic and nonphotosynthetic), which starts the 3rd d after the beginning of imbibition. Transcription from the leaf-/cotyledon-specific P1 promoter of the rpI21 gene starts on the first imbibition day. Inhibition of chloroplast biogenesis by bleaching in the presence of norflurazon has no influence on the expression from this P1 promoter, suggesting that the onset of transcription of nuclear gene rpI21 is independent of a plastid signal.

Base Sequence↗
PubMed1995

Effects of light and chloroplast functional state on expression of nuclear genes encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase in long hypocotyl (hy) mutants and wild-type Arabidopsis thaliana.

In a previous study of Arabidopsis thaliana (J. Dewdney, T.R. Conley, M.-C. Shih, H.M. Goodman [1993] Plant Physiol 103: 1115-1121), it was postulated that both blue light receptor- and phytochrome-mediated pathways contribute to regulation of the nuclear genes encoding A and B subunits of glyceraldehyde-3-phosphate dehydrogenase (GAPA and GAPB). Here were report on the involvement of a nuclear gene encoding a putative blue-light receptor (HY4) and of a nuclear gene encoding phytochrome A apoprotein (PHYA) in regulation of the GAPA and GAPB genes in response to blue and far-red light. Continuous light irradiation experiments with the hy4 mutant demonstrate that the HY4 gene product is required for full expression of GAPA, GAPB, and one or more of the nuclear genes encoding small subunits of of ribulose-1,5-bisphosphate carboxylase/oxygenase. Continuous light irradiation and fluence-response studies with the phyA-101 mutant show that phytochrome A functions in far-red light regulation of GAPA, GAPB, nuclear genes encoding small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase, and CAB genes. Phytochromes A and B alone either do not participate in red light-mediated gene regulation or have redundant functions, as shown by analysis of phyA-101 and phyB-1 single mutants. In addition, the hypothesis that chloroplast-nucleus interactions affect GAPA and GAPB gene regulation was tested. Herbicide-mediated photooxidative damage to chloroplasts in A thaliana seedlings strongly decreased the maximum amount of GAPA and GAPB steady-state mRNA detected in continuous-light irradiation experiments. Full expression of the GAPB genes is dependent on the presence of functional chloroplasts.

Arabidopsis↗
PubMed1984

Protein synthesis during chloroplast development in Spirodela oligorhiza. Coordinated synthesis of chloroplast-encoded and nuclear-encoded subunits of ATPase and ribulose-1,5-bisphosphate carboxylase.

We have studied qualitative and quantitative changes of several parameters during chloroplast development in Spirodela oligorhiza (duckweed). On a dry weight basis, the amount of protein increases from 2.5% (w/w) in dark-grown to 7.8% (w/w) in light-grown fronds. At the same time the amount of starch drops from 50% to 27% (w/w). Using an immunochemical quantification method we have found that during greening of etiolated plants the amount of all subunits of the ATPase complex per frond increases 10-fold, whereas the level of the subunits of ribulose-1,5-biphosphate carboxylase increases 50-fold. Cytochrome f was found to be present in dark-grown Spirodela and the amount of this polypeptide per frond increases about 30-fold. The concentration of a polypeptide that possibly represents a cytochrome b6 subunit increases about 10-fold upon greening. The molar ratio of the CF1-beta and CF1-gamma subunits of the ATPase complex varies over 2-3, while in all stages of chloroplast development studied the molar ratio of the carboxylase subunits is about 1. As these values are in agreement with the stoichiometrical amounts in the native protein complexes, we conclude that the synthesis of CF1-beta and CF1-gamma, as well as the synthesis of the large and small carboxylase subunits, are strictly coordinated during chloroplast biogenesis in Spirodela oligorhiza.

Adenosine Triphosphatases↗
PubMed1994

Chloroplast protein import. Chloroplast envelopes and thylakoids have different abilities to unfold proteins.

Proteins have to be at least partially unfolded upon passage through the biological membranes. Previous studies with a dihydrofolate reductase fusion protein containing a chloroplast transit peptide showed that stabilization of the tertiary structure of the fusion protein by binding of a ligand, methotrexate, failed to block its translocation across the envelopes, suggesting that chloroplast envelopes have strong activity to unfold proteins [America, T., Hageman, J., Guéra, A., Rook, F., Archer, K., Keegstra, K. & Weisbeek, P. (1994) Plant Mol. Biol. 24, 283-294]. In the present study, we have analyzed in vitro translocation of a fusion protein consisting of the entire plastocyanin precursor and dihydrofolate reductase across the chloroplast envelope membranes and the thylakoid membrane. In the presence of methotrexate, the fusion protein was imported into the stroma but its translocation across the thylakoid membrane was blocked. The fusion protein that bound to the envelope became susceptible to digestion by thermolysin. These results suggest that, while the envelope membranes can unfold the methotrexate-bound fusion protein to allow its passage, the thylakoid membrane cannot unfold the fusion protein that has re-bound to methotrexate in the stroma.

Amino Acid Sequence↗
PubMed1991

Specific binding of chloroplast proteins in vitro to the 3' untranslated region of spinach chloroplast petD mRNA.

A detailed analysis of RNA-protein complex formation in the 3' untranslated region of spinach chloroplast petD mRNA has been carried out. Five chloroplast proteins that interact with petD RNA in this region, which contains an inverted repeat sequence capable of forming a hairpin structure, have been identified. A 33-kDa protein recognizes specifically the double-stranded stem of the hairpin structure; mutations that disrupt base pairing at the base of the stem reduce or eliminate protein binding. A 57-kDa protein recognizes specifically an AU-rich sequence motif that is highly conserved in petD genes of different higher plant species. The 57-kDa protein and possibly the 33-kDa protein form stable complexes with petD RNA in vitro and may interact with each other. In addition, their interaction with petD RNA is highly sensitive to heparin. The three other proteins, of 100, 32, and 28 kDa, display little sequence or structural binding specificity apart from their preference for uridine-rich sequences. They also interact with the 3' untranslated regions of other chloroplast RNAs such as those of psbA and rbcL. The functions of these proteins in the regulation of petD gene expression, including possible roles in transcription termination and RNA stability, are discussed.

Base Composition↗
PubMed1999

Mutations altering the predicted secondary structure of a chloroplast 5' untranslated region affect its physical and biochemical properties as well as its ability to promote translation of reporter mRNAs both in the Chlamydomonas reinhardtii chloroplast and in Escherichia coli.

Random mutations were generated in the sequence for the 5' untranslated region (5'UTR) of the Chlamydomonas reinhardtii chloroplast rps7 mRNA by PCR, the coding sequence for the mutant leaders fused upstream of the lacZ' reporter in pUC18, and transformed into Escherichia coli, and white colonies were selected. Twelve single base pair changes were found at different positions in the rps7 5'UTR in 207 white colonies examined. Seven of the 12 mutant leaders allowed accumulation of abundant lacZ' message. These mutant rps7 leaders were ligated into an aadA expression cassette and transformed into the chloroplast of C. reinhardtii and into E. coli. In vivo spectinomycin-resistant growth rates and in vitro aminoglycoside adenyltransferase enzyme activity varied considerably between different mutants but were remarkably similar for a given mutant expressed in the Chlamydomonas chloroplast and in E. coli. The variable effect of the mutants on aadA reporter expression and their complete abolition of lacZ' reporter expression in E. coli suggests differences in the interaction between the 5'UTR of rps7 and aadA or lacZ' coding regions. Several rps7 5'UTR mutations affected the predicted folding pattern of the 5'UTR by weakening the stability of stem structures. Site-directed secondary mutations generated to restore these structures in the second stem suppressed the loss of reporter activity caused by the original mutations. Additional site-directed mutations that were predicted to further strengthen (A-U-->G-C) or weaken (G-C-->A-U) the second stem of the rps7 leader both resulted in reduced reporter expression. This genetic evidence combined with differences between mutant and wild-type UV melting profiles and RNase T1 protection gel shifts further indicate that the predicted wild-type folding pattern in the 5'UTR is likely to play an essential role in translation initiation.

5' Untranslated Regions↗
PubMed2004

Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development.

The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development.

Amino Acid Sequence↗
PubMed1992

Variations in chloroplast proteins and nucleotide sequences of three chloroplast genes in Triticum and Aegilops.

Two alloplasmic wheat lines having the same common wheat nucleus but the cytoplasms of Aegilops crassa and Ae. columnaris together with the corresponding normal line (control) were used in the two-dimensional gel electrophoresis of soluble and thylakoid membrane proteins of the chloroplast. Three chloroplast polypeptides: the Rubisco large subunit, the beta subunit of ATP synthase, and an unidentified 31 kDa protein, differed in the common wheat and two Aegilops cytoplasms. Three chloroplast genes, atpB, atpE and trnM, that respectively encode the beta and epsilon subunits of ATP synthase and tRNA(met), were sequenced. The atpB gene differed by two synonymous base substitutions, whereas the other two genes were identical in the two Aegilops cytoplasms. From the predicted amino acid sequences, the beta subunits of the ATP synthase in the Aegilops cytoplasms were assumed to have three amino acid substitutions: Ala by Val, Asp- by Ala, and Gln by Lys+, in contrast to the cytoplasm of common wheat. This accounts for the difference in pI values found for the common wheat and Aegilops cytoplasms. The two base substitutions for the atpE genes of common wheat and the Aegilops cytoplasms were synonymous. The differences detected in the genes encoding the two subunits of ATP synthase do not appear to be ascribable to the differences in phenotypic effects for the common wheat and Aegilops cytoplasms. The base substitution rate of the atpB-atpE-trnM gene cluster was similar to that of the rbcL gene. From the rate for the atpB gene alone, evolutionary divergence of the wheat-Aegilops complex is assumed to have begun ca. 3.0 x 10(6) years ago, as compared to ca. 8.0 x 10(6) years ago for the divergence of the wheat-Aegilops complex and barley.

Amino Acid Sequence↗
PubMed1991

Acyl carrier protein import into chloroplasts. Both the precursor and mature forms are substrates for phosphopantetheine attachment by a soluble chloroplast holo-acyl carrier protein synthase.

Recently a chloroplast holo-acyl carrier protein (holoACP) synthase activity was identified which attached the phosphopantetheine prosthetic group to acyl carrier protein, producing holoACP (Fernandez and Lamppa (1990) Plant Cell 2, 195-206). Here we show that the mature form of ACP (apoACP), after entry into the chloroplast and removal of the transit peptide, is a substrate for modification by the holoACP synthase. Modification occurs optimally at 37 degrees C and is inhibited by 5 mM 3',5'-ADP and 2 mM EDTA. An ACP construct (matACP) lacking the transit peptide was also converted to the holoACP form in an organelle-free assay, independent of precursor cleavage. The matACP construct was used to monitor the chromatographic separation of the holoACP synthase from the transit peptidase. Superose 12 gel filtration analysis indicates that the holoACP synthase has an apparent Mr of approximately 50,000. Using fractions enriched for the holoACP synthase it was demonstrated that the precursor of ACP is also modified in the presence of CoA and subsequently can be proteolytically processed directly to holoACP. Kinetic analysis, however, indicates that removal of the transit peptide is a much faster reaction than phosphopantetheine addition, suggesting that apoACP is the primary substrate for the chloroplast holoACP synthase in vivo.

Acyl Carrier Protein↗
PubMed1979

[Chloroplast DNA cloning in Escherichia coli. II. The properties of the recombinant plasmids bearing the EcoRI fragments of pea chloroplast DNA and the cloning of the DNA sequences with rRNA genes].

Previously a method of selection of colicine-defective recombinant plasmids by mitomycin C was described. A series of recombinant plasmids (CPS) with various EcoRI-fragments of pea chloroplast DNA has been obtained. This paper describes some properties of cloned fragments replicated in Escherichia coli. The alkali stability of recombinant plasmid DNAs has been demonstrated, indicating the absence of ribonucleotides in their structure. Heterogeneity of chloroplast DNA in nucleotide composition was demonstrated using ultracentrifugation analysis of CPS-plasmid DNAs in CsCl-actinomycin D density gradient. Pea chloroplast rDNA was cloned in recombinant plasmids.

Base Sequence↗
PubMed1993

Import and assembly of the beta-subunit of chloroplast coupling factor 1 (CF1) into isolated intact chloroplasts.

The transit peptide gene of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Nicotiana plumbaginifolia was fused to the tentoxin-resistant beta-subunit gene of chloroplast coupling factor 1 (CF1) from Nicotiana tabacum via a linker sequence. The consequent fusion gene encodes the entire gene sequences of both the transit peptide and the beta-subunit of CF1 without a single change of amino acid residues. The fusion gene was in vitro expressed in a coupled transcription-translation system as a 62-kDa precursor and was imported into isolated intact chloroplasts of Nicotiana longiflora. The imported precursor was found to be processed to the expected mature beta-subunit size. Evidence is presented that radioactively labeled beta-subunit was incorporated into mature CF1 and not just nonspecifically associated with the thylakoid membranes. Since the fusion protein containing only the transit peptide sequence was imported and apparently correctly processed, it may not be necessary to include N-terminal amino acids of the mature small subunit for correct proteolytic cleavage in the chloroplast stroma.

Amino Acid Sequence↗
PubMed1980

Preferential transcription of cloned maize chloroplast DNA sequences by maize chloroplast RNA polymerase.

Zea mays chloroplast DNA-dependent RNA polymerase in vitro preferentially transcribes maize chloroplast DNA sequences incorporated in cloned chimeric bacterial plasmids. Preferential transcription is dependent on the presence of a 27.5-kilodalton polypeptide, the S factor, which has been purified from maize chloroplasts, and also on the template's being in the supercoiled form.

Journal Article↗
PubMed1991

Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii.

Chloroplast transformation of Chlamydomonas reinhardtii has been accomplished by agitating cell wall-deficient cells in the presence of glass beads and DNA. By using the atpB gene as the selected marker and cells grown in 0.5 mM 5-fluorodeoxyuridine, we have recovered up to 50 transformants per microgram of DNA. This method is easy and does not require specialized equipment, although it is not as efficient as the tungsten particle bombardment method [Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johnson, A. M., Jones, A. R., Randolph-Anderson, B. L., Robertson, D., Klein, T. M., Shark, K. B. & Sanford, J. C. (1988) Science 240, 1534-1537]. By using particle bombardment, we have developed a cotransformation approach in which spectinomycin-resistant 16S rRNA-encoding DNA is the selected marker, and we have demonstrated that cotransformation of an unselected marker on an independent replicon is very efficient. We have used this strategy (i) to recover transformants with partially deleted atpB genes that could not otherwise have been selected since they did not restore photosynthetic capability to a recipient carrying a more extensive atpB deletion and (ii) to generate specific deletion mutations in a wild-type recipient. This methodology should allow the introduction of any desired change into the chloroplast genome, even in the absence of phenotypic selection, and thus a detailed functional analysis of any chloroplast DNA sequence should be possible.

Journal Article↗