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Chloroplast and cytoplasmic enzymes: isolation and sequencing of cDNAs coding for two distinct pea chloroplast aldolases.

Two cDNAs which correspond to two very similar Class I aldolases have been isolated from a pea (Pisum sativum L.) cDNA library. With the exception of one codon they match the experimentally determined N-terminal sequence of a pea chloroplast aldolase. The deduced C-terminal sequence of one of these clones is unique among Class I aldolases. The deduced C-terminus of the other is more like the C-terminus of other eucaryotic Class I aldolases. Comparisons of sequence homology suggest that the pea chloroplast isozymes are only marginally more closely related to the anaerobically induced plant aldolases than to aldolases from animals.

Amino Acid Sequence↗

The effect of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide on calcium binding and associated changes in chloroplast structure and chlorophyll a fluorescence in spinach chloroplasts.

1. Chemical modification of carboxyl groups on the chloroplast membrane with a water-soluble carbodiimide plus a nucleophile caused inhibition of Ca-2plus binding. 2. Both binding sites were affected and showed a decrease in the number of binding sites and an increase in the dissociation constant. 3. Cation-induced changes in chlorophyll a fluorescence and structural changes (deltaA540) were inhibited at the same carbodiimide concentrations as Ca-2plus binding, emphasizing the relationship between these processes. 4. Chloroplasts that were illuminated with high intensity light for short time periods showed a decrease in the carbodiimide-mediated inhibition of Ca-2plus binding.

Calcium↗

Iodination of chloroplasts. I. Properties of iodinated chloroplasts.

Spinach chloroplasts exposed to iodide can be washed free of the bulk of the iodide. In the presence of lactoperoxidase and H2O2, iodide can be introduced into chloroplasts in high amounts and in non diffusible forms. The resultant particles, which have been named iodochloroplasts, extrude their iodide upon stimulation by light. The form and the amount of extruded iodide bears a definite relationship to the amount of incident light. A flash of marginally effective light is additive to the next such flash even after a lapse of 10 min of darkness. These and other properties of iodochloroplasts may make them of great use in the study of intermediate reactions of photosynthesis.

Biological Transport↗

Characterisation of a chloroplast-encoded secY homologue and atpH from a chromophytic alga. Evidence for a novel chloroplast genome organisation.

secY is a prokaryotic gene that encodes the SecY protein, an integral membrane component of the prokaryotic protein translocation apparatus. A chloroplast-encoded secY homologue has been identified in the unicellular, chromophytic alga, Pavlova lutherii. The gene predicts a protein composed of ten membrane-spanning regions, that is approximately 25% homologous and 50% similar to bacterial and plastid SecY proteins. The secY gene from P. lutherii is independent of the ribosomal protein (rp) gene cluster to which it is closely linked in other organisms. In P. lutherii secY is located 5' to atpI and atpH. Since, in higher plants the atpIHFA gene cluster and the rp gene cluster are separated by approximately 50 kb, we conclude, this indicates a novel chloroplast gene arrangement in P. lutherii.

Amino Acid Sequence↗

Chloroplast DNA deletions associated with wheat plants regenerated from pollen: possible basis for maternal inheritance of chloroplasts.

Albinism in plants regenerated from pollen by anther culture may result from the same mechanisms as postulated to be responsible for maternal inheritance of chloroplasts. Consistent with this view, Southern blotting was used to show that large regions of the chloroplast genome (over 80% in one case) were deleted in the major ctDNA molecules present in eight albino wheat plants regenerated from separate pollen calluses. A wide variety of deleted ctDNA molecules was present in these plants. Most albino plants appeared to contain a heterogeneous population of ctDNA molecules. Albino plant 6 contained one major type of deleted ctDNA molecule that had a circular 39 kb restriction map. The retention of only two Pst l (unaltered in size) fragments in albino plant 7 may delimit a region of ctDNA that contains a replication origin.

Base Sequence↗

Stimulation of a Chlamydomonas chloroplast promoter by novobiocin in situ and in E. coli implies regulation by torsional stress in the chloroplast DNA.

We have characterized regulation of a complex Chlamydomonas reinhardtii chloroplast (PA) whose activity is stimulated by the DNA gyrase inhibitor novobiocin, both in the alga itself and in a heterologous Escherichia coli plasmid system. Since novobiocin is known to reduce torsional stress in E. coli DNA, we interpret our results to mean that PA is regulated by torsional stress in the chloroplast DNA. In E. coli, where we could readily manipulate PA, we found that this regulation depends on sequences upstream of PA. These sequences contain at least two different kinds of silencing elements that inhibit PA in the absence of novobiocin. Novobiocin stimulates PA only when the promoter-distal silencing element is present.

Base Sequence↗

Chloroplast biosystematics: chloroplast DNA as a molecular probe.

The classification of plants has traditionally been dependent upon the comparative analysis of morphological and biochemical data. In this paper the use of molecular probe analysis of chloroplast DNA (ctDNA) is used to expand the data base used in taxonomic studies. Chloroplast DNA size, homogeneity, the global arrangement of ctDNA structure, gene content, gene cluster array and gene sequence determination are discussed as useful criteria in the analysis of phylogenetic relationships.

Base Sequence↗

The Shine-Dalgarno-like sequence is a negative regulatory element for translation of tobacco chloroplast rps2 mRNA: an additional mechanism for translational control in chloroplasts.

Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation. Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.

5' Untranslated Regions↗

The complete chloroplast genome sequence of Pelargonium x hortorum: organization and evolution of the largest and most highly rearranged chloroplast genome of land plants.

The chloroplast genome of Pelargonium x hortorum has been completely sequenced. It maps as a circular molecule of 217,942 bp and is both the largest and most rearranged land plant chloroplast genome yet sequenced. It features 2 copies of a greatly expanded inverted repeat (IR) of 75,741 bp each and, consequently, diminished single-copy regions of 59,710 and 6,750 bp. Despite the increase in size and complexity of the genome, the gene content is similar to that of other angiosperms, with the exceptions of a large number of pseudogenes, the recognition of 2 open reading frames (ORF56 and ORF42) in the trnA intron with similarities to previously identified mitochondrial products (ACRS and pvs-trnA), the losses of accD and trnT-ggu and, in particular, the presence of a highly divergent set of rpoA-like ORFs rather than a single, easily recognized gene for rpoA. The 3-fold expansion of the IR (relative to most angiosperms) accounts for most of the size increase of the genome, but an additional 10% of the size increase is related to the large number of repeats found. The Pelargonium genome contains 35 times as many 31 bp or larger repeats than the unrearranged genome of Spinacia. Most of these repeats occur near the rearrangement hotspots, and 2 different associations of repeats are localized in these regions. These associations are characterized by full or partial duplications of several genes, most of which appear to be nonfunctional copies or pseudogenes. These duplications may also be linked to the disruption of at least 1 but possibly 2 or 3 operons. We propose simple models that account for the major rearrangements with a minimum of 8 IR boundary changes and 12 inversions in addition to several insertions of duplicated sequence.

Chloroplasts↗

Nucleotide sequences of three soybean chloroplast tRNAsLeu and re-examination of bean chloroplast tRNA2Leu sequence.

The nucleotide sequences of soybean chloroplast tRNAsLeu were determined using post-labeling techniques. Comparison of the primary structures of soybean chloroplast tRNAsLeu with their bean, maize and spinach counterparts only show few base differences. Contrary to previously published results (1) a re-examination of bean tRNALeu sequence shows that this tRNA resembles soybean and maize tRNA2Leu in structure.

Base Sequence↗

Acyl carrier protein (ACP) import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holo-ACP synthase.

Import of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP from back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.

Acyl Carrier Protein↗

Chloroplast elongation factors are synthesized in the chloroplast.

The elongation factor G (EF-Gchl) and elongation factor Tu (EF-Tuchl) present in spinach chloroplasts become labelled when isolated chloroplasts are incubated in the light with radioactive methionine. EF-Gchl and EF-Tuchl account for approximately 0.04% and 0.2% respectively of the total radioactivity incorporated by isolated organelles.

Chloroplasts↗

ATP synthesis and hydrolysis in chloroplast membranes. Differential inhibition by antibodies to chloroplast coupling factor 1.

1. Divalent antibodies against chloroplast coupling factor 1 inhibited the factor ATPase, ATP synthesis, hydrolysis and Pi-ATP exchange in chloroplasts. These antibodies also inhibited coupled electron flow rates but not the basal or uncoupled rates. 2. Several types of non-precipitating, modified antibodies prepared from the original antibody preparation strongly inhibited the ATPase and Pi-ATP exchange reaction but had little effect on ATP formation. 3. It is suggested that the inhibition of ATP synthesis by the divalent antibodies is probably due to an indirect blocking of the active site, while the inhibition of ATP-utilizing reactions by the modified antibodies is related to their effect on the transfer of ATP from a non-catalytic to a catalytic site on coupling factor 1, via an energy-dependent conformational change.

Adenosine Triphosphate↗

Transport of proteins into chloroplasts. Partial purification of a chloroplast protease involved in the processing of important precursor polypeptides.

We have partially purified a soluble protease from Pisum sativum chloroplasts involved in the processing of precursor polypeptides imported into the organelle. The enzyme processes precursors of both stromal and thylakoid proteins to the mature size, but is inactive against all proteins so far tested other than precursors destined for the chloroplast. The enzyme processes precursors from wheat and barley, and is therefore not species-specific. It has a relative molecular mass of about 180 000 and a pH optimum near 9. The enzyme is inhibited by ethylenediamine tetraacetate and 1,10-phenanthroline but not by serine- or thiol-protease inhibitors.

Biological Transport↗

Chloroplast ATPase in Acetabularia acetabulum: purification and characterization of chloroplast F1-ATPase.

ATPases were isolated from chloroplasts of the unicellular marine alga Acetabularia acetabulum. Two preparations of ATPase, a chloroplast-enriched fraction and an alpha beta gamma-complex were compared. The alpha beta gamma-complex was released into an EDTA solution and purified by anion-exchange chromatography, hydrophobic chromatography, and gel permeation chromatography. The subunit composition of this enzyme appeared to be 52-53 (alpha), 51 (beta), and 40 (gamma) kDa from SDS-PAGE. ATPase activity was enriched about 260-fold to a specific activity of approximate 4.1 U.mg protein-1. The catalytic properties of the alpha beta gamma-complex were as follows: pH optimum at 7.5; substrate specificity, ATP > ITP, GTP > UTP = CTP (Km for ATP 0.2 mM); divalent cation requirement, Mg2+ = Mn2+ = Co2+ > Zn2+ > Ni2+ > Ca2+; ATPase activity was inhibited by monovalent anions (NO3-, SCN-), while monovalent cations had neither inhibitory nor stimulatory effect. Orthovanadate had no inhibitory effect on the enzyme activity of alpha beta gamma-complex. Azide was the most effective inhibitor of the alpha beta gamma-complex. N-Terminal amino acid sequences of the alpha and beta subunits were not obtained and appeared to be blocked. The gamma subunit gave a sequence of AGLKEMKD-XIGSVXNTKKI, which showed 60% similarity to the gamma subunits of spinach and Chlamydomonas reinhardtii CF1-ATPase and EF1-ATPase.

Acetabularia↗

Interconversion of two distinct states of active CF0-CF1 (chloroplast ATPase complex) in chloroplasts.

Chloroplast ATPase complex is activated by illumination in the presence or absence of dithiothreitol. ATPase complex which has been activated without dithiothreitol catalyzes ATP hydrolysis which is insensitive to stimulation by NH4Cl and is highly sensitive to medium pH. Addition of dithiothreitol during illumination results in an increase in the stimulating effect of NH4Cl on ATP hydrolysis and a decrease in pH sensitivity of ATP hydrolysis. With increasing time in the dark, the ability of NH4Cl to stimulate ATP hydrolysis decreases and the effect of pH on the ATP hydrolysis increases. The onset of resistance to NH4Cl stimulation and the increase in sensitivity to pH are accelerated by ADP and the acceleration is inhibited by Pi. ATP hydrolysis restores NH4Cl sensitivity and renders the activity more resistant to pH. These results suggest that active chloroplast ATPase complex converts its state reversibly from the NH4Cl-insensitive and highly pH-sensitive one to the NH4Cl-sensitive and relatively pH-insensitive one. The conversion from the former to the latter requires both sulfhydryl compound and energy.

Ammonium Chloride↗

[Cloning chloroplast DNA in escherichia coli. I. Construction and selection of recombinant plasmids containing fragments of pea chloroplast DNA].

Fragments produced by digestion of Pisum sativum chloroplast DNA with EcoRI were examined by agarose gel electrophoresis. These EcoRI-fragments were joined in vitro to Apr-ColE1 RSF2124 plasmid and cloned in Escherichia coli. Methods of molecular cloning of plasmid chimeras by success gradient centrifugation and repeated transformation and selection of recombinant plasmids using mytomicin C were used for cloning hybrid plasmids with various EcoRI fragments of pea chloroplast DNA has been obtained.

Chloroplasts↗

Chloroplasts in envelopes: CO(2) fixation by fully functional intact chloroplasts.

Dan Arnon, Bob Whatley, Mary Belle Allen, and their colleagues, were the first to obtain evidence for 'complete photosynthesis by isolated chloroplasts' albeit at rates which were 1% or less of those displayed by the intact leaf. By the 1960s, partly in the hope of confirming full functionality, there was a perceived need to raise these rates to the same order of magnitude as those displayed by the parent tissue. A nominal figure of 100 mumol/mgcchlorophyll/h (CO(2) assimilated or O(2) evolved) became a target much sought after. This article describes the contributions that Dick Jensen and Al Bassham [(1966) Proc Natl Acad Sci USA 56: 1095-1101], and my colleagues and I, made to the achievement of this goal and the way in which it led to a better understanding of the role of inorganic phosphate in its relation to the movement of metabolites across chloroplast envelopes.

Journal Article↗