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PubMed1981

[Light-induced generation of membrane potential in intact chloroplasts suspended in H2O or D2O media].

The membrane potential changes induced by short flashes and continuous light were investigated in isolated chloroplasts of Peperomia metallica suspended in H2O- or D2O media. The potential generated in H2O-suspended chloroplasts by a single turnover flash is approximately two times lower than the maximal level of potential induced by continuous light. The photoelectric response of D2O-suspended chloroplasts differs from that of H2O-suspended chloroplasts by an increased amplitude and a prolonged phase of the potential rise. Te dark decay of the potential proceeds 2-3 times slower in the D2O-suspended chloroplasts as compared to the H2O-suspended chloroplasts. The magnitude of the flash-induced potential is somewhat lower for the chloroplasts in D2O than for the chloroplasts in the H2O medium. The results obtained suggest that the substitution of H2O for D2O results in a decrease of the ionic conductance and an increase of stability of thylakoid membranes. It was shown that the rise of electrical potential under continuous illumination proceeds in two stages. The difference kinetics of membrane potential changes are observed under conditions of separate activity of two systems of photosynthesis.

Chloroplasts↗

PubMedDate not supplied

[Kinetics and mechanism of inactivation of isolated chloroplasts].

A detailed kinetic analysis has been performed of a multistep inactivation of chloroplasts. The kinetic model suggested involves the formation of chloroplast forms differing in stability and activity. A comparison of the kinetic model with the experimental data shows that the mechanism of inactivation of isolated pea chloroplasts consists of at least two forms displaying different activity in the Hill reaction and different stability in solution. The effect was studied of the nature of the buffer and destruction products on the kinetics of chloroplast inactivation in the process of "ageing". In phosphate buffer, where the concentration of phosphate exceeds 40 mM, the effect of the destruction products of chloroplasts on their inactivation is insignificant. The pH dependence on the inactivation kinetics suggests that the pH region from 6 to 9 affects only the rapid kinetic process resulting in the increase in the chloroplast activity; irreversible inactivation of chloroplasts is pH-independent. The temperature dependence of the irreversible inactivation kinetics has been studied and the activation parametres of this stage have been determined. Possible molecular mechanism of the limiting stages of the inactivation of isolated chloroplasts are discussed, which can explain the kinetic data obtained.

Chloroplasts↗

PubMed1976

Transfer of substrates across the chloroplast envelope.

The chloroplast represents a relatively autonomous metabolic compartment within the plant cell. It is surrounded by an envelope consisting of two membranes of which the inner membrane is the functional barrier. Utilizing the energy of light the chloroplast is able to synthesize dihydroxyacetonephosphate from carbon dioxide and water. To provide the cell with this substrate, inorganic phosphate is required. In the case of phosphate deficiency the product of CO2 fixation may be temporarily stored within the chloroplast as starch. Specific transport processes across the inner envelope membrane permit the transfer of metabolites between the chloroplast and the cytosol. The phosphate translocator facilitates the export of dihydroxyacetone phosphate in exchange for inorganic phosphate. It also catalyzes a shuttle for inorganic phosphate with 3-phosphoglycerate, permitting the indirect transfer of reducing equivalents and of ATP from the chloroplast to the cytosol. The dicarboxylate carrier transporting various dicarboxylates may be suited for the transfer of reducing equivalents from the cytosol into the chloroplast. The ATP translocator, catalyzing a transport of ATP into the chloroplast in exchange for ADP, appears to be important for providing the chloroplast with ATP during the night phase, as required for the mobilization of starch.

Biological Transport↗

PubMed1994

Recognition of novel and divergent higher plant chloroplast ribosomal proteins by Escherichia coli ribosome during in vivo assembly.

Architecture of higher plant chloroplast ribosomes involves additional protein domains over that found in the Escherichia coli ribosome, although the rRNAs in these two kinds of ribosomes are very similar in length and sequence (Subramanian, A. R. (1993) Trends Biochem. Sci. 18, 177-180). Here, we show that two chloroplast-specific protein domains (a novel chloroplast ribosomal protein of the 30 S subunit, called Psrp-1 or S22, and a divergent protein of the 50 S subunit with long terminal extensions and low homology to its E. coli counterpart, L21) are both incorporated in E. coli ribosomes and polysomes when their gene constructs are expressed in E. coli. Also, the 67-residue NH2-terminal extension in chloroplast L21 by itself is incorporated. Thus, our results indicate preexisting binding sites for novel chloroplast-specific ribosomal proteins/domains on eubacterial ribosomes. Additionally, we observed cleavage of the chloroplast-targeting transit peptide (present in the expressed Psrp-1 precursor), indicating protease(s) of the required specificity in E. coli cells. The expression of chloroplast L21 with its NH2-terminal extension was inhibitory to E. coli growth, suggesting a drastic effect of the latter on some property of L21. Expression of Psrp-1 was neutral, consistent with a function only in chloroplast translation. Based on analysis of the assembly of Psrp-1 and various L21 fragments in E. coli ribosomes, a general model for studying ribosomal protein-ribosome interactions is suggested.

Base Sequence↗

PubMed1994

Cloning and characterization of chloroplast and cytosolic forms of cyclophilin from Arabidopsis thaliana.

Cyclophilin (CyP), a protein with peptidyl-prolyl cis-trans isomerase (rotamase) activity, is the specific cellular target of cyclosporin A. We have isolated cDNA clones of two genes (designated ROC1 and ROC4) encoding CyP homologs from Arabidopsis thaliana (L.). The protein products of these genes are distinct from a previously identified Arabidopsis CyP. ROC1 is expressed in all tested plant organs and encodes a protein which is highly similar to previously described cytosolic CyP isoforms of other plants. In contrast, ROC4 is expressed only in photosynthetic organs and encodes a protein which includes an amino-terminal extension with properties of known chloroplast transit peptides. In vitro import experiments using the putative precursor protein to ROC4 showed that the protein is imported into chloroplasts where it is processed to the predicted mature size. Rotamase assays and immunoblot analysis of subcellular fractions indicate the presence of a CyP isoform in the stroma of chloroplasts but not in the thylakoid membranes or thylakoid lumen. Together, these data show that ROC4 is a novel CyP isoform which is located in the stroma of chloroplasts. In vitro chloroplast import of precursors of other chloroplast proteins was unaffected by concentrations of cyclosporin A which completely inhibit rotamase activity of chloroplast stromal CyP. Thus, this activity is not essential for protein import into chloroplasts.

Amino Acid Isomerases↗

PubMed2026

Chloroplast Haplotype Analysis Reveals High Genetic Similarity Among Central Asian Prunus Species.

Genetic variation in four wild Prunus taxa (P. fruticosa, P. erythrocarpa, P. verrucosa and P. griffithii var. tianshanica) was investigated for the first time using six chloroplast DNA regions (matK, r rpl16, ycf1_1, ycf1_2, ndhF and trnH-psbA) analysed through CAPS-based SNP detection. The results revealed weak chloroplast differentiation among P. erythrocarpa, P. verrucosa and P. griffithii var. tianshanica. However, chloroplast variation exhibited a strong geographic signal across the studied populations. The observed chloroplast variation primarily reflected geographic structuring rather than clear differentiation among these closely related taxa. In contrast, P. fruticosa showed distinct chloroplast haplotypes not shared with the other taxa. These findings demonstrate that the developed chloroplast CAPS marker system is effective for detecting chloroplast haplotype variation but has limited discriminatory power among closely related wild Prunus taxa. Further studies using nuclear markers and genome-wide approaches will be required to better resolve their genetic relationships and evolutionary history.

Haplotypes↗

PubMed1996

Post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii.

The biosynthesis of the photosynthetic apparatus depends on the concerted action of the nuclear and chloroplast genetic systems. Numerous nuclear and chloroplast mutants of Chlamydomonas deficient in photosynthetic activity have been isolated and characterized. While several of these mutations alter the genes of components of the photosynthetic complexes, a large number of the mutations affect the expression of chloroplast genes involved in photosynthesis. Most of these mutations are nuclear and only affect the expression of a single chloroplast gene. The mutations examined appear to act principally at post-transcriptional steps such as RNA stability, RNA processing, cis- and trans-splicing and translation. Directed chloroplast DNA surgery through biolistic transformation has provided a powerful tool for identifying important cis elements involved in chloroplast gene expression. Insertion of chimeric genes consisting of chloroplast regulatory regions fused to reporter genes into the chloroplast genome has led to the identification of target sites of the nuclear-encoded functions affected in some of the mutants. Biochemical studies have identified a set of RNA-binding proteins that interact with the 5'-untranslated regions of plastid mRNAs. The binding activity of some of these factors appears to be modulated by light and by the growth conditions.

Animals↗

PubMed2002

Cadmium accumulation in the chloroplast of Euglena gracilis.

Intracellular distribution of Cd, cysteine, glutathione, and Cd-induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 mM CdCl2, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll-purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd2+, since uptake of added CdCl2 in isolated chloroplasts exhibited a hyperbolic type of kinetics with a Km of 57 &mgr;M and Vmax of 3.7 nmol (mg protein)-1 min-1. The contents of cysteine and glutathione markedly increased in both chloroplasts (7-19 times) and cytosol (4-9 times) by exposure to Cd2+, although they were always higher in the cytosol. Thiol-containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol-peptides.

Journal Article↗

PubMed1999

Light perception and the role of the xanthophyll cycle in blue-light-dependent chloroplast movements in lemna trisulca L

In most higher plants, chloroplasts move towards the periclinal cell walls in weak blue light (WBL) to increase light harvesting for photosynthesis, and towards the anticlinal walls as an escape reaction, thus avoiding photo-damage in strong blue light (SBL). The photo- receptor(s) triggering these responses have not yet been identified. In this study, the role of zeaxanthin as a blue-light photoreceptor in chloroplast movements was investigated. Time-lapse 3D confocal imaging in Lemna trisulca showed that individual chloroplasts responded to local illumination when one half of the cell was treated with light of different intensity or spectral quality to that received by the other half, or was maintained in darkness. Thus the complete signal perception, transduction and effector system has a high degree of spatial resolution and is consistent with localization of part of the transduction chain in the chloroplasts. Turnover of xanthophylls was determined using HPLC, and a parallel increase was observed between zeaxanthin and chloroplast movements in SBL. Ascorbate stimulated both a transient increase in zeaxanthin levels and chloroplast movement to profile in physiological darkness. Conversely, dithiothreitol blocked zeaxanthin production and responses to SBL and, to a lesser extent, WBL. Norflurazon preferentially inhibited SBL-dependent chloroplast movements. Increases in zeaxanthin were also observed in strong red light (SRL) when no directional chloroplast movements occurred. Thus it appears that a combination of zeaxanthin and blue light is required to trigger responses. Blue light can cause cis-trans isomerization of xanthophylls, thus photo-isomerization may be a critical link in the signal transduction pathway.

Journal Article↗

PubMed1976

Transcriptional origin of Euglena chloroplast tRNAs.

tRNA.DNA hybridization studies indicate that Euglena chloroplast tRNAs are transcriptional products of the chloroplast genome, which contains approximately 26 tRNA cistrons. Hybridization with purified chloroplast tRNA(Phe) and tRNA(Asp) shows that the chloroplast genome contains one cistron for each of these two species. No hybridization of chloroplast tRNA with nuclear DNA was observed. tRNAs from Euglena cytoplasm, Escherichia coli, and Agmenellum quadraduplicatum do not compete with chloroplast tRNA for hybridization with chloroplast DNA. Evidence is presented that photoinduction of chloroplast tRNAs is at the level of transcription rather than maturation of tRNA precursor molecules.

Journal Article↗

PubMed1989

Seasonal variation in the photosynthetic capacity of a willow (Salix cv. Aquatica gigantea) canopy. 2 Comparison of the structure and function of chloroplasts at different levels in the canopy.

Development of leaf chloroplast ultrastructure at five levels in a willow (Salix cv. Aquatica gigantea) canopy was followed during one growing season in the field. Changes in chloroplast ultrastructure were compared with the rate of CO(2) uptake of the same leaves. The highest rates of CO(2) uptake were recorded in young leaves exposed to full available sunlight. In these leaves, the area of the grana stacks was less than 20% of the total chloroplast area and the degree of thylakoid stacking was less than 1.5. The chloroplasts of these leaves contained large amounts of starch and small amounts of plastoglobuli. As the canopy grew and the leaves in the lower parts of the canopy became shaded, the structure of the chloroplast thylakoids gradually changed. In leaves at the two lowest levels of the canopy, the degree of stacking at the end of the growing season was close to 2 and correspondingly the rate of CO(2) uptake was low. The areas of grana stacks and plastoglobuli in these chloroplasts increased and were about 30 and 10% of the chloroplast area, respectively, by the end of the growing season. The increase in the degree of thylakoid stacking was caused by increased biosynthesis of grana lamellae, which in general were thinner than the lamellae of young leaves. The length of the stroma lamellae did not change with leaf age. Morphometric measurements showed that the structure of the chloroplasts in leaves 160 cm above ground was dynamic and responsive to environmental conditions so that photosynthetic capacity remained high for 7 weeks despite an increase in leaf shading.

Journal Article↗

PubMed1992

Flow cytometry of spinach chloroplasts : determination of intactness and lectin-binding properties of the envelope and the thylakoid membranes.

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly "intact" and "disrupted" chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes.

Journal Article↗

PubMed1994

Carbonic Anhydrase Activity in Isolated Chloroplasts of Wild-Type and High-CO2-Dependent Mutants of Chlamydomonas reinhardtii as Studied by a New Assay.

In an assay of carbonic anhydrase (CA), NAH14CO3 soltution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment. The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C. reinhardtii had 740 [plus or minus] 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 [plus or minus] 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 [plus or minus] 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw-15, believed to lack an internal CA, had 2.8 [plus or minus] 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 [plus or minus] 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 [mu]M inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80[deg]C for 10 min inhibited this CA activity by 90.8 [plus or minus] 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity.

Journal Article↗

PubMed1994

Expression of the Arabidopsis Gene Akr Coincides with Chloroplast Development.

Reduced expression of a nuclear gene of Arabidopsis thaliana, Akr, results in the formation of chlorotic plants due to a block in the proplastid-to-chloroplast development pathway (H. Zhang, D.C. Scheirer, W. Fowle, H.M. Goodman [1992] Plant Cell 4: 1575-1588). In an effort to discern the function of the Akr gene product in chloroplast development, transgenic plants containing an Akr::[beta]-glucuronidase gene fusion were constructed to monitor the spatial and temporal patterns of Akr expression. Akr is expressed only in chloroplast-containing tissues and maximal expression occurs during the seedling stage, coincident with chloroplast development. This result is consistent with the hypothesis that Akr is required at an early stage of chloroplast development. The effects of an AKR deficiency on the expression of nuclear and plastid genes required for photosynthetic activity were also examined. Within chloroplast-deficient leaves of plants in which Akr expression is limited by the presence of Akr antisense transgenes or truncated Akr sense transgenes, mRNAs for the nuclear genes Cab2, Cab4, RbcS, and GapA are present at wild-type levels; similarly, levels of mRNAs for the plastid genes rbcL and psbA are not affected by the AKR deficiency. Thus, although expression of these photosynthetic genes is tightly coordinated with the development and maintenance of chloroplasts in wild-type plants, their expression is unaffected in AKR-deficient chlorotic leaves. Therefore, we propose that Akr functions in a pathway different from the one controlling the expression and regulation of the photosynthetic genes during chloroplast development, and at a specific developmental stage after the putative plastid factor is made.

Journal Article↗

PubMed1972

Light-stimulated Production of a Chloroplast-localized System for Protein Synthesis in Euglena gracilis.

Chloroplasts and proplastids isolated respectively from autotrophic and dark-adapted cells of Euglena gracilis strain Z incorporated (14)C-l-leucine into protein. In each case the incorporation was inhibited by chloramphenicol (50% inhibition at about 5 mug/ml for chloroplasts and 30 mug/ml for proplastids), but not appreciably by cycloheximide at concentrations up to 200 mug/ml. Chloroplasts from autotrophic cells incorporated leucine into protein at rates of about 10 pg leucine per mg RNA in one minute, but isolated proplastids were only 5 to 10% as active. When dark-adapted cells were illuminated there was little increase in the activity of the chloroplast fraction during the first 12 hr. Between 12 and 24 hr, when there was a rapid increase in the rate of synthesis of chlorophyll, the capacity of the chloroplast fraction for protein synthesis increased markedly. Suppression of the formation of a chloroplast-localized system for protein synthesis by treating the cells with chloramphenicol and the lack of such an effect with cycloheximide suggests that certain of the proteins which form part of a functional chloroplast system for protein synthesis are themselves synthesized within the chloroplasts.

Journal Article↗

PubMed1973

Chloroplast Response to Low Leaf Water Potentials: II. Role of Osmotic Potential.

Electron transport in chloroplasts isolated from desiccated sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves was compared with electron transport in sunflower chloroplasts in sorbitol-containing media having various osmotic potentials. In media having low osmotic potentials and dichloroindophenol as electron acceptor, the activity for electron transport was inhibited, but the inhibition was much less than that due to comparable desiccation in vivo. The inhibition at low osmotic potentials was rapidly reversed by returning the chloroplasts to media having high osmotic potentials, but the activity of chloroplasts from desiccated tissue showed no reversal when the chloroplasts were placed in media having high osmotic potentials. Nevertheless, the inhibition of chloroplast activity due to desiccation in vivo was basically reversible, because chloroplasts recovered quickly when they were rehydrated in vivo. The large differences between desiccation in vivo and exposure to low osmotic potential in vivo indicate that osmotic solutions did not reproduce the effects of tissue desiccation. It is concluded that decreases in the Gibbs free energy of water due to decreased osmotic potentials probably have only a small effect on electron transport in chloroplasts from desiccated tissue and do not account for the major effects of leaf desiccation on electron transport.

Journal Article↗

PubMed1974

Nitrite assimilation and amino nitrogen synthesis in isolated spinach chloroplasts.

The assimilation of nitrite leading to de novo synthesis of amino nitrogen in a chloroplast-enriched fraction isolated from freshly harvested young spinach (Spinacia oleracea L.) leaves was demonstrated. The preparations showed approximately 55% intact chloroplasts as determined by light scattering properties and fixed CO(2) at rates of approximately 100 mumoles hr(-1) mg chlorophyll(-1).The chloroplast-enriched fraction contained the enzymes, nitrite reductase and NADPH-glutamate dehydrogenase, needed for the reduction of nitrite and incorporation of ammonia into glutamate. Kinetic studies showed that the reduction of nitrite by the chloroplast-enriched fraction is light-dependent, and the process proceeds at rates of 6 to 12 mumoles hr(-1) mg chlorophyll(-1). The addition of nitrite to the chloroplast preparation caused a 3-fold increase in the production of alpha-amino nitrogen when compared with the control without nitrite. There was a stoichiometric relation between amino-nitrogen synthesis and nitrite disappearance from the medium. The ratio of amino-nitrogen: NO(2) (-) ranged from 0.6 to 0.9. The initial rate of amino-nitrogen production was faster when (alpha)-ketoglutarate was added to the nitrite reducing chloroplast medium than when it was omitted. However, these high rates were not sustained and the total amino-nitrogen production at the end of a 30-minute period was only slightly higher. These data show that chloroplasts are functionally able and contain the enzyme complement necessary to utilize light energy for the reduction of nitrite to amino nitrogen. Thus, chloroplasts should be considered as a major site for in vivo amino-nitrogen synthesis in green plants.

Journal Article↗

PubMed1975

Photosynthetic activity and membrane polypeptide composition of supergranal chloroplasts from plant tissue cultures containing a viruslike particle.

Tissue culture cells of Streptanthus tortuosus (Kell.) var. orbiculatus (Greene) Hall (Cruciferae), having a viruslike particle in their nucleoli, the STV cell line, contain "supergranal" chloroplasts. Freeze-fracture studies of chloroplasts of a control cell line, which lacks the viruslike particles, reveal two complementary faces similar to those observed in spinach chloroplasts. Replicas of freeze-fractured STV supergranal chloroplasts, however, show that one membrane face (B) contains widely spaced 80 A particles and the other face (C) is essentially smooth. Isolated STV supergranal chloroplasts lack photosystem II activity as indicated by their inability to reduce dichlorophenolindophenol and are unable to reduce NADP with electrons from photosystem II or from ascorbate-reduced dichlorophenolindophenol. However, partial photosystem I activity is indicated by the reduction of methyl viologen with electrons from dichlorophenolindophenol-ascorbate. This supports the concept that there is not a direct correspondence between grana formation and photosystem II activity. Electrophoresis shows that all of the major polypeptide bands present in the STV supergranal chloroplasts are also present in the control chloroplast membranes. One band, molecular weight 33,000, is present in a greatly increased amount in the STV supergranal chloroplast membranes and may be associated with grana stacking.

Journal Article↗