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PubMed1997

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts.

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin (oriA). Transformants were recovered only with the plasmid containing oriA, and all transformants contained an integrated plasmid copy at oriA, suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.

Animals↗

PubMed2002

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts.

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.

Blotting, Western↗

PubMed2001

Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas.

By studying the import of radioactively labelled small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into chloroplasts of the green alga C. reinhardtii cw-15 protein delivery to chloroplasts was found to vary during the cell cycle. Chloroplasts were isolated from highly synchronous cultures at different time points during the cell cycle. When pSS was imported into 'young' chloroplasts isolated early in the light period about three times less pSS was processed to small subunit SS than in 'mature' chloroplasts from the middle of the light period. In 'young' chloroplasts also, less pSS was bound to the envelope surface. During the second half of the light period the import competence of isolated chloroplasts decreased again when based on chlorophyll content or cell volume, but did not change significantly when related to chloroplast number. Measurements of pSS binding to the surface of chloroplasts of different age indicated that the adaptation of protein import competence during the cell cycle is due to a variation of the number of binding sites per chloroplast surface area, rather than to modulation of the binding constant.

Adenosine Triphosphate↗

PubMed1979

Mutations in nine chloroplast loci of Chlamydomonas affecting different photosynthetic functions.

Chloroplast components known to be coded by chloroplast DNA include chloroplast rRNAs, tRNAs, and the large subunit of ribulose-bisphosphate carboxylase. Because these components comprise less than 3% of the estimated coding capacity of the chloroplast genome, most chloroplast gene functions have yet to be identified. One approach to this problem is the isolation and characterization of mutations in the chloroplast genome affecting specific photosynthetic functions. Recently we have found that such mutations can be preferentially recovered by using arsenate selection on cells previously grown in 5-fluorodeoxyuridine. Sixteen mutants thus isolated have been localized into nine chloroplast loci, based on their ability to recombine and produce photosynthetically competent progeny. Mutants at two loci show the characteristic syndrome of photosynthetic defects that results from a deficiency in chloroplast protein synthesis. These have been found to lack chloroplast ribosome monomers. Mutants at three loci are missing chlorophyll-protein complex I in their thylakoid membranes. Mutants at three other loci are deficient in membrane polypeptides known to be associated with the chloroplast coupling factor.

Alleles↗

PubMed1965

Light-induced volume changes in spinach chloroplasts.

A light-dependent mechanism that results in a slow, high-amplitude swelling of spinach chloroplasts in vitro has been discovered. The swelling is readily observed by optical and gravimetric methods, and by the use of an electronic particle counter; all show a 100 per cent increase of chloroplast volume in the light with an approximately 10-minute half-time. The existence of an osmotic mechanism for chloroplast swelling in the dark is confirmed. The volume of illuminated chloroplasts versus NaCl concentration represents the addition of osmotic and light effects. The action of light is enhanced by electron flow cofactors, such as phenazine methosulfate (PMS). However, neither conditions for ATP hydrolysis or synthesis nor NH(4)Cl influence the time course and extent of swelling. Hence, high-amplitude chloroplast swelling is light- (or electron flow), but not energy-dependent. A remarkable inhibitory effect of inorganic phosphate on chloroplast swelling is observed in the light, but not in the dark. Another action of light on chloroplasts is known to result in a shrinkage of chloroplasts which is rapid, reversible, energy-dependent, and requires phosphate. Thus phosphate determines the action of light on chloroplast volume. Since shrinkage is reversible, but swelling is not, it may be that they reflect physiological and deteriorative processes, respectively. Chloroplasts and mitochondria appear to control their volume by similar mechanisms.

Adenosine Triphosphate↗

PubMed1973

Autoradiographic evidence for many segregating DNA molecules in the chloroplast of Ochromonas danica.

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-(3)H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.

Autoradiography↗

PubMed2003

Chloroplast redox control of nuclear gene expression--a new class of plastid signals in interorganellar communication.

Chloroplasts are genetically semiautonomous organelles that contain their own subset of 100-120 genes coding for chloroplast proteins, tRNAs, and rRNAs. However, the great majority of the chloroplast proteins are encoded in the nucleus and must be imported into the organelle after their translation in the cytosol. This arrangement requires a high degree of coordination between the gene expression machineries in chloroplasts and nucleus, which is achieved by a permanent exchange of information between both compartments. The existence of such coordinating signals has long been known; however, the underlying molecular mechanisms and signaling routes are not understood. The present data indicate that the expression of nuclear-encoded chloroplast proteins is coupled to the functional state of the chloroplasts. Photosynthesis, which is the major function of chloroplasts, plays a crucial role in this context. Changes in the reduction/oxidation (redox) state of components of the photosynthetic machinery act as signals, which regulate the expression of chloroplast proteins in both chloroplasts and nucleus and help to coordinate the expression both in compartments. Recent advances in understanding chloroplast redox regulation of nuclear gene expression are summarized, and the importance for intracellular signaling is discussed.

Cell Nucleus↗

PubMed2000

Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by microbeam irradiation.

Chloroplast relocation in mesophyll cells of Arabidopsis thaliana was observed microscopically and analyzed by microbeam irradiation. Chloroplasts located along the anticlinal walls in dark-adapted cells. When part of a cell was irradiated with a microbeam of high fluence rate blue light (B) simultaneously with background red light (R) on the whole cell, the chloroplasts moved towards the B-irradiated area, but did not enter the beam. The background R illumination activated cytoplasmic motility as well as chloroplast movement. Without R illumination, there was little chloroplast relocation. In light-adapted cells in which the chloroplasts were spread over the cell surface perpendicular to the incident light, R-illumination had the same effect. Under background R, the chloroplasts moved out of the area irradiated with a B microbeam of 8 or 30 W m(-2) (avoidance response), but chloroplasts outside the beam moved towards the area irradiated with the B microbeam (accumulation response). These results suggest that the signals for accumulation and avoidance responses were generated in a single cell by high fluence rate B. cry1cry2, npq1 and nph1 mutants showed B-induced chloroplast relocation. Both the accumulation and avoidance responses were observed in all the mutants, although in the nph1 mutant, the sensitivity of accumulation movement was slightly lower than that of the wild type. We discuss the possible photoreceptor for B-induced chloroplast relocation.

Adaptation, Physiological↗

PubMed2004

ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase.

The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.

Amino Acid Motifs↗

PubMed1994

Acyl carrier protein (ACP) import into chloroplasts. Covalent modification by a stromal holoACP synthase is stimulated by exogenously added CoA and inhibited by adenosine 3',5'-bisphosphate.

During the import of the precursor for the acyl carrier protein (ACP) into chloroplasts, apoACP is converted to holoACP by the attachment of a phosphopantetheine group transferred from coenzyme A (CoA) by a chloroplast holoACP synthase [Fernandez, M. and Lamppa, G. (1990) Acyl carrier protein import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holoACP synthase, Plant Cell 2, 195-206]. Here it is shown that exogenous addition of CoA to intact chloroplasts in the import assay stimulates the conversion of apoACP to holoACP. If adenosine 3',5'-bisphosphate [Ado(3',5')P2], the byproduct of the transfer reaction, was also included the extent of conversion was greatly reduced. CoA has its effect after ACP precursor (pre-ACP) import and proteolytic removal of the transit peptide, thus indicating that the chloroplast holoACP synthase resides in the stroma where fatty acid synthase is found. When Ado(3',5')P2 was added alone to the import assay, it inhibited the synthesis of holoACP. Inhibition of the conversion of apo- to holoACP with Ado(3',5')P2 made it possible to examine whether the holoform of preACP could be imported into chloroplasts. Pre-apoACP was synthesized in Escherichia coli and shown to be competent for import in an ATP- and temperature-dependent manner. A partially purified chloroplast holoACP synthase converted 60-90% of the pre-apoACP to pre-holoACP. Pre-holoACP incubated with chloroplasts in the presence of Ado(3',5')P2 yielded > 60% holoACP, whereas the control reaction with pre-apoACP gave primarily apoACP. Hence the phosphopantetheine prosthetic group of ACP does not block precursor movement through the translocation apparatus of the chloroplast envelope.

Acyl Carrier Protein↗

PubMed1980

Pigment mutants of Chlamydomonas with deformed plastids and increased levels of chloroplast nucleic acids.

The chloroplast ultrastructure of two Chlamydomonas reinhardtii pigment variant mutants, U3N and U3A, is strikingly different from that of the wild type. The mutant chloroplast has greatly lowered levels of chlorophyll a and b, and lacks the usual ordered thylakoid membrane structure. The amount of chloroplast ribosomes is increased, but the pyrenoid and surrounding starch grains appear to be unaltered. Our biochemical analyses have shown that, while the properties of chloroplast DNA, ribosomal RNA, and ribosomes in these mutants appeared to be normal, their relative amounts per cell increased markedly when compared to the wild type. In U3N these increases were approximately 60% for chloroplast DNA and 80% for chloroplast ribosomes. However, the ratio of chloroplast rDNA genes to total chloroplast DNA remained unchanged as shown by DNA-rRNA hybrdization. We propose that (1) The enhanced level of chloroplast ribosomes in these mutants is a direct consequence of the elevated amount of chloroplast DNA. Both of these increases may, in turn, arise from defective mechanism for their control. (2) These mutants grow successfully in the absence of functional photosynthesis, provided an external carbon source is available to them, but functional plastid DNA, ribosomes and protein synthesis may still be a requirement for normal starch metabolism.

Chlamydomonas↗

PubMed2006

Complete nucleotide sequence of the cotton (Gossypium barbadense L.) chloroplast genome with a comparative analysis of sequences among 9 dicot plants.

Recently, the complete chloroplast genome sequences of many important crop plants were determined, and this can be considered a major step forward toward exploiting the usefulness of chloroplast genetic engineering technology. Economically, cotton is one of the most important crop plants for many countries. To further our understanding of this important crop, we determined the complete nucleotide sequence of the chloroplast genome from cotton (Gossypium barbadense L.). The chloroplast genome of cotton is 160,317 base pairs (bp) in length, and is composed of a large single copy (LSC) of 88,841 bp, a small single copy (SSC) of 20,294 bp, and two identical inverted repeat (IR) regions of 25,591 bp each. The genome contains 114 unique genes, of which 17 genes are duplicated in the IRs. In addition, many open reading frames (ORFs) and hypothetical chloroplast reading frames (ycfs) with unknown functions were deduced. Compared to the chloroplast genomes from 8 other dicot plants, the cotton chloroplast genome showed a high degree of similarity of the overall structure, gene organization, and gene content. Furthermore, the sequences of the genes showed high degrees of identity at the DNA and amino acid levels. The cotton chloroplast genome was somewhat longer than the chloroplast genomes of most of the other dicot plants compared here. However, this elongation of the cotton chloroplast genome was found to be due mainly to expansions of the intergenic regions and introns (non-coding DNA). Moreover, these expansions occurred predominantly in the LSC and SSC regions.

Base Sequence↗

PubMedDate not supplied

The effect of an antiserum to plastocyanin on various chloroplast preparations.

A monospecific antiserum to tobacco plastocyanin agglutinates stroma-free swellable chloroplasts from wild type tobacco, (Nicotiana tobacum var. John William's Broadleaf) from the tobacco aurea mutant Su/su2, (Nicotiana tabacum var. Su/su2) from Antirrhinum majus and spinach (Spinacia oleracea). In this condition the antiserum inhibits linear photosynthetic electron flow in tobacco and spinach chloroplasts. This inhibition of electron transport as well as the agglutination are not observed if the chloroplasts have been sonicated prior to antiserum addition. This is due to the fact that plastocyanin is removed by ultasonication. The antiserum stimulates a number of photophosphorylation reactions in tobacco chloroplasts. This stimulation is always larger in the aurea mutant chloroplasts from yellow leaf patches of a variegated tobacco mutant (N. tabacum, var. NC 95) than in the green type chloroplasts. The stimulation appears to be a consequence of the inhibition of linear electron transport. The antiserum does not affect PMS-mediated cyclic photophosphorylation in tobacco chloroplasts from the wild type whereas the reaction appears stimulated in the tobacco mutant chloroplasts. However, menadione-mediated cyclic photophosphorylation is inhibited upon addition of the antiserum. The same is true for noncyclic photophosphorylation coupled to electron transport in the aerobic system diaminodurene/ascorbate leads to methylviologen in the presence of N-tetraphenyl-p-phenylenediamine in spinach chlorplasts. If the lamellar system of Antirrhinum and spinach has lost is swellability neither agglutination nor inhibition of electron transport is observed. However, also in this state antibodies to plastocyanin are specifically adsorbed onto the surface to the thylakoid membrane. This state which is characterized by a morphologically well preserved lamellar system is realized in chloroplast preparations from Antirrhinum and spinach and is termed stroma-freed chloroplasts. In both states of the molecular structure of the thylakoid membrane, plastocyanin is located in the outer surface of the thylakoid. However, it cannot be excluded that functioning plastocyanin is also located in the interior of the thylakoid membrane.

2,6-Dichloroindophenol↗

PubMed2001

The hypocotyl chloroplast plays a role in phototropic bending of Arabidopsis seedlings: developmental and genetic evidence.

Chloroplasts of guard cells and coleoptiles have been implicated in the sensory transduction of blue light. The present study was aimed at establishing whether the chloroplast of the hypocotyl from Arabidopsis, another blue light-responding organ, has similar characteristics to that of sensory-transducing guard cell and coleoptile chloroplasts. Results showed that the phototropic curvature and arch length induced by blue light in Arabidopsis seedlings matched the distribution of mature chloroplasts in the bending hypocotyl. The bending arch consistently included the region of the hypocotyl containing mature chloroplasts, and never extended beyond that region. Manipulation of the extent of greening of dark-grown hypocotyls by varying red light pretreatments elicited blue light-stimulated curvatures and arch lengths that depended on the duration of the red light pretreatment and on the distribution of mature chloroplasts in the hypocotyl. Albino psd2 mutants of Arabidopsis, which lack mature chloroplasts, are devoid of phototropic sensitivity under conditions in which wild-type seedlings show large curvatures. The star mutant of Arabidopsis has a delayed greening and a delayed phototropic response as compared with wild type. Measurements of photosynthetic oxygen evolution and carbon fixation, dark respiration, and light-dependent zeaxanthin formation in the hypocotyl showed features similar to those of guard cells and coleoptiles, and distinctly different from those of mesophyll tissue. These results indicate that the hypocotyl chloroplast has characteristics similar to those associated with guard cell and coleoptile chloroplasts, and that phototropic bending of Arabidopsis hypocotyls appears to require mature chloroplasts.

Arabidopsis↗

PubMed1986

Motility in the siphonous green alga Bryopsis. II. Chloroplast movement requires organized arrays of both microtubules and actin filaments.

The cortical cytoplasm of the giant cells of Bryopsis contains hundreds of interconnected microtubule (MT) bundles aligned along the cell's long axis. Actin fibers show an extensive but not exclusive superposition with these MT bundles. Chloroplasts move parallel to the bundles. Colchicine (0.5 mM), vinblastine (0.1 mM), and the herbicide ami-prophosmethyl (APM, 1-5 microM) strongly inhibit chloroplast movement and severely disrupt both the MT and the actin network. Additionally, APM leads to the appearance of large actin bundles up to 5 microns in diameter and several tens of micron in length. Erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA, 1 mM) does not block chloroplast movement, but affects chloroplast behavior by causing transient aggregations. The MT network is not significantly changed by EHNA, but actin fibers converge in large, radially symmetric complexes in regions of chloroplast aggregates. Cytochalasin D (CD, 1-10 micrograms/ml) leads to a significant but transient reduction of chloroplast speed within the first 60 min, as the actin network breaks down into small foci. Within the next 1 to 3 h of treatment, these foci segregate into massive clusters where chloroplasts remain immobilized. At the same time, chloroplast movement recovers in other areas of the cell. This recovery coincides with the reappearance of actin filament bundles in these cell regions. The MT cytoskeleton is not significantly affected by CD. These data are inconsistent with a mechanism of chloroplast movement in Bryopsis based solely on either MTs or actin, but instead they suggest an intimate interaction of both cytoskeletal networks in maintaining the spatial organization of the cytoplasm and in supporting chloroplast movement.

Actin Cytoskeleton↗

PubMed1985

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi.

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45 degrees C induces the expression of 10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36 degrees C to 42 degrees C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)RNAs endure in the cells at 42 degrees C up to 5 h but are no longer translated in vivo, whereas some poly(A)RNAs persist and are translated. As in pea, a poly(A)RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)RNA coding for the 22-kd HSP appears after 1 h at 36 degrees C. Its synthesis increases with the temperature of incubation up to 42 degrees C, although it decreases after 2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26 degrees C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.

Journal Article↗

PubMed2002

Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species.

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H2O2 level and membrane lipid peroxidation was observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione-S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non-specific peroxidases (POD) were found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt-stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt-treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt-treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt-dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre-exposure of Lpa plants to salt-treatment conferred cross-tolerance to paraquat-induced oxidative stress while increased oxidative damage by paraquat-treatment was found in salt-stressed Lem plants.

Journal Article↗

PubMed1989

Tobacco mosaic virus RNA enters chloroplasts in vivo.

Several lines of evidence are presented to allow us to conclude that tobacco mosaic virus (TMV) RNA enters the chloroplast in vivo. Chloroplasts were prepared from either directly inoculated or systemically infected leaves of tobacco plants inoculated with one of several strains of the virus and from uninfected control plants. Intact chloroplasts were isolated on Percoll gradients and treated with pancreatic RNase and thermolysin to destroy potential TMV virions and RNA on the outside or bound to their surfaces. Northern blot analysis of RNA extracted from these chloroplasts demonstrated that full-length TMV RNA was present within the chloroplasts prepared from both directly inoculated and systemically invaded leaves. Only genomic length, but not subgenomic length, RNA was found in the chloroplast extracts, indicating a selectivity of the transport of the viral RNA into the chloroplast. A temperature-sensitive TMV mutant (Ts 38), in which no virions are formed at 35 degrees C, was used to demonstrate that at that restrictive temperature viral RNA is detected in the chloroplast, indicating that free viral RNA can enter the chloroplast rather than intact virions. To our knowledge, the transport of a foreign RNA species into chloroplasts has not been reported previously.

Journal Article↗