Chloroplast Replication Research Articles

Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana.

Plastids in vascular plants have various differentiated forms, among which amyloplasts are crucial for starch storage and plant productivity. Despite the vast knowledge of the binary-fission mode of chloroplast division, our understanding of the replication of non-photosynthetic plastids, including amyloplasts, remains limited. Recent studies have suggested the involvement of stromules (stroma-filled tubules) in plastid replication when the division apparatus is faulty. However, details of the underlying mechanism(s) and their relevance to normal processes have yet to be elucidated. Here, we developed a live analysis system for studying amyloplast replication using Arabidopsis (Arabidopsis thaliana) ovule integuments. We showed the full sequence of amyloplast development and demonstrated that wild-type amyloplasts adopt three modes of replication, binary fission, multiple fission, and stromule-mediated fission, via multi-way placement of the FtsZ ring. The minE mutant, with severely inhibited chloroplast division, showed marked heterogeneity in amyloplast size, caused by size-dependent but wild-type modes of plastid fission. The dynamic properties of stromules distinguish the wild-type and minE phenotypes. In minE cells, extended stromules from giant amyloplasts acquired stability, allowing FtsZ ring assembly and constriction, as well as the growth of starch grains therein. Despite hyper-stromule formation, amyloplasts did not proliferate in the ftsZ null mutant. These data clarify the differences between amyloplast and chloroplast replication and demonstrate that the structural plasticity of amyloplasts underlies the multiplicity of their replication processes. Furthermore, this study shows that stromules can generate daughter plastids via the assembly of the FtsZ ring.

Structural and functional insights into the chloroplast division site regulators PARC6 and PDV1 in the intermembrane space

Chloroplast division involves the coordination of protein complexes from the stroma to the cytosol. The Min system of chloroplasts includes multiple stromal proteins that regulate the positioning of the division site. The outer envelope protein PLASTID DIVISION1 (PDV1) was previously reported to recruit the cytosolic chloroplast division protein ACCUMULATION AND REPLICATION OF CHLOROPLAST5 (ARC5). However, we show here that PDV1 is also important for the stability of the inner envelope chloroplast division protein PARALOG OF ARC6 (PARC6), a component of the Min system. We solved the structure of both the C-terminal domain of PARC6 and its complex with the C terminus of PDV1. The formation of an intramolecular disulfide bond within PARC6 under oxidized conditions prevents its interaction with PDV1. Interestingly, this disulfide bond can be reduced by light in planta, thus promoting PDV1-PARC6 interaction and chloroplast division. Interaction with PDV1 can induce the dimerization of PARC6, which is important for chloroplast division. Magnesium ions, whose concentration in chloroplasts increases upon light exposure, also promote the PARC6 dimerization. This study highlights the multilayer regulation of the PDV1-PARC6 interaction as well as chloroplast division.

Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement.

Viral replication and movement are intimately linked; however, the molecular mechanisms regulating the transition between replication and subsequent movement remain largely unknown. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein promotes viral replication and movement by interacting with the αa replicase and TGB1 movement proteins. Here, we found that γb is palmitoylated at Cys-10, Cys-19, and Cys-60 in Nicotiana benthamiana, which supports BSMV infection. Intriguingly, non-palmitoylated γb is anchored to chloroplast replication sites and enhances BSMV replication, whereas palmitoylated γb protein recruits TGB1 to the chloroplasts and forms viral replication-movement intermediate complexes. At the late stages of replication, γb interacts with NbPAT15 and NbPAT21 and is palmitoylated at the chloroplast periphery, thereby shifting viral replication to intracellular and intercellular movement. We also show that palmitoylated γb promotes virus cell-to-cell movement by interacting with NbREM1 to inhibit callose deposition at the plasmodesmata. Altogether, our experiments reveal a model whereby palmitoylation of γb directs a dynamic switch between BSMV replication and movement events during infection.

Moonlighting proteins: putting the spotlight on enzymes

ABSTRACTAROGENATE DEHAYDRATASE2 (ADT2) is a member of the Arabidopsis thaliana ADT family. All members of this family act as arogenate dehydratases in phenylalanine biosynthesis, decarboxylating/dehydrating arogenate to phenylalanine. ADT2 is detected in stromules, and as a ring around the equatorial plane of dividing chloroplasts, indicating it has a second, non-enzymatic function in chloroplast division. Here, we provide further evidence for this alternative role of ADT2. First, we demonstrate that ADT2 and FtsZ co-localize around the equatorial plane at the same time. Second, FtsZ expression in an adt2 mutant was analyzed, as well as ADT2 expression in three Arabidopsis chloroplast division mutants, ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), ARC5 and ARC6. In arc3 and arc6 mutants, ADT2 is misexpressed and resembles the expression of FtsZ in the same mutants. However, in the arc5 mutant, ADT2 ring positioning is observed at constriction points indicating proper relative timing. ADT2 expression in the arc mutants shows that the role of ADT2 in chloroplast division occurs prior to ARC5, but is dependent on ARC3 and ARC6.Abbreviations used: ADT: arogenate dehydratase, ARC: accumulation and replication of chloroplasts, CFP: cyan fluorescent protein, dpi: days post infiltration, FtsZ: filamentous temperature sensitive Z, PD: plastid division, Phe: phenylalanine, YFP: yellow fluorescent protein.

Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana.

Summary Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity.To characterize the transcript level component of metabolic regulation, genome‐wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time‐course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterized alongside lipid accumulation.Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes.Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal‐derived biofuels.

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best-characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild-type poplar hybrid Populus tremula x P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding gamma-glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild-type plants; soil contamination significantly decreased biomass accumulation in both wild-type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two-dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild-type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal-contaminated field sites. Further experiments are required to test these assumptions.

Nucleotide Depletion and Chloroplast Division

We have described the identification of crinkled leaves 8 (cls8) which contains a mutation within the gene encoding the large subunit of ribonucleotide reductase (RNR), the enzyme that catalyses the rate limiting step in the synthesis of deoxyribonucleotide triphosphates (dNTPs) for DNA synthesis and repair. The mutation resulted in plants with altered leaf and flower morphology, reduced root growth, bleached leaf sectors and reduced levels of dNTPs. An interesting consequence of the mutation was its effect on chloroplast division. Mutant plants had fewer, larger chloroplasts and a reduced number of chloroplast genomes compared to wild type plants. The morphological phenotype may be a consequence of altered chloroplast replication.

Regulation of Brassica rapa chloroplast proliferation in vivo and in cultured leaf disks.

To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differential stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differential phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.

A family of selfish minicircular chromosomes with jumbled chloroplast gene fragments from a dinoflagellate.

Chloroplast genes of several dinoflagellate species are located on unigenic DNA minicircular chromosomes. We have now completely sequenced five aberrant minicircular chromosomes from the dinoflagellate Heterocapsa triquetra. These probably nonfunctional DNA circles lack complete genes, with each being composed of several short fragments of two or three different chloroplast genes and a common conserved region with a tripartite 9G-9A-9G core like the putative replicon origin of functional single-gene circular chloroplast chromosomes. Their sequences imply that all five circles evolved by differential deletions and duplications from common ancestral circles bearing fragments of four genes: psbA, psbC, 16S rRNA, and 23S rRNA. It appears that recombination between separate unigenic chromosomes initially gave intermediate heterodimers, which were subsequently stabilized by deletions that included part or all of one putative replicon origin. We suggest that homologous recombination at the 9G-9A-9G core regions produced a psbA/psbC heterodimer which generated two distinct chimeric circles by differential deletions and duplications. A 23S/16S rRNA heterodimer more likely formed by illegitimate recombination between 16S and 23S rRNA genes. Homologous recombination between the 9G-9A-9G core regions of both heterodimers and additional differential deletions and duplications could then have yielded the other three circles. Near identity of the gene fragments and 9G-9A-9G cores, despite diverging adjacent regions, may be maintained by gene conversion. The conserved organization of the 9G-9A-9G cores alone favors the idea that they are replicon origins and suggests that they may enable the aberrant minicircles to parasitize the chloroplast's replication machinery as selfish circles.

Leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth.

Brassica napus L. (oilseed rape) was grown from seeds on a reconstituted soil contaminated with cadmium (100 mg Cd kg-1 dry soil), resulting in a marked chlorosis of the leaves which was investigated using a combination of biochemical, biophysical and physiological methods. Spectroscopic and chromatographic analyses of the photosynthetic pigments indicated that chlorosis was not due to a direct interaction of Cd with the chlorophyll biosynthesis pathway. In addition, mineral deficiency and oxidative stress were apparently not involved in the pigment loss. Leaf chlorosis was attributable to a marked decrease in the chloroplast density caused by a reduction in the number of chloroplasts per cell and a change in cell size, suggesting that Cd interfered with chloroplast replication and cell division. Relatively little Cd was found in the chloroplasts and the properties of the photosynthetic apparatus (electron transport, protein composition, chlorophyll antenna size, chloroplast ultrastructure) were not affected appreciably in plants grown on Cd-polluted soil. Depth profiling of photosynthetic pigments by phase-resolved photoacoustic spectroscopy revealed that the Cd-induced decrease in pigment content was very pronounced at the leaf surface (stomatal guard cells) compared to the leaf interior (mesophyll). This observation was consistent with light transmission and fluorescence microscopy analyses, which revealed that stomata density in the epidermis was noticeably reduced in Cd-exposed leaves. Concomitantly, the stomatal conductance estimated from gas-exchange measurements was strongly reduced with Cd. When plants were grown in a high-CO2 atmosphere (4,000 microliters CO2 l-1), the inhibitory effect of Cd on growth was not cancelled, suggesting that the reduced availability of CO2 at the chloroplast level associated with the low stomatal conductance was not the main component of Cd toxicity in oilseed rape.

Study on replication and morphology of chloroplasts in Arabidopsis thaliana

In higher plants, only a few proplastids in shoot meristem cells undergo replication to develop into a large number of chloroplasts in mesophyll cells. The developmental process accompanies morphological changes of the organelle; small (~1 mm) and amorphous proplastids shape into large (4-6 mm) and uniformly spherical chloroplasts. In the present study, we analyzed evolutionary conserved nuclear genes of Arabidopsis thaliana that encode chloroplast proteins involved in chloroplast replication and morphology. FtsZ, a bacterial homolog of tubulins, forms a ring at midpoint of dividing chloroplasts as well as bacterial cells. Recent studies hypothesized that one family (FtsZ1) members of higher plant FtsZ proteins target into chloroplasts while another family (FtsZ2) members localize in cytoplasm. Contrary to this hypothesis, FtsZ2 fused with green fluorescent protein (GFP) was shown to target into chloroplasts by confocal laser scanning microscopy (CLSM). The FtsZ2-GFP distributed as fibers and dots that formed network structures within chloroplasts. Moreover, we identified a new nuclear gene from Arabidopsis that shows homology to eubacterial cell division site-determining factor MinE. GFP experiments demonstrated that MinE is also a chloroplast stromal protein. Transgenic Arabidopsis plants overexpressing MinE caused aberrant chloroplast replication and morphology, indicating that MinE is the third conserved factor involved in chloroplast division. Furthermore, we observed by CLSM that root leucoplasts frequently emanate tubular structures, called stromules. Some stromules connected with other leucoplasts while some extended towards cell nuclei. We are currently studying the effects of altered expression of chloroplast division genes on stromule proliferation.

Fine mapping of replication origins (ori A and ori B) in Nicotiana tabacum chloroplast DNA.

Using a partially purified replication complex from tobacco chloroplasts, replication origins have been localized to minimal sequences of 82 (pKN8, positions 137 683-137 764) and 243 bp (pKN3, positions 130 513-130 755) for ori A and ori B respectively. Analysis of in vitro replication products by two-dimensional agarose gel electrophoresis showed simple Y patterns for single ori sequence-containing clones, indicative of rolling circle replication. Double Y patterns were observed when a chloroplast DNA template containing both ori s (pKN9) was tested. Dpn I analysis and control assays with Escherichia coli DNA polymerase provide a clear method to distinguish between true replication and DNA repair synthesis. These controls also support the reliability of this in vitro chloroplast DNA replication system. EM analysis of in vitro replicated products showed rolling circle replication intermediates for single ori clones (ori A or ori B), whereas D loops were observed for a clone (pKN9) containing both ori s. The minimal ori regions contain sequences which are capable of forming stem-loop structures with relatively high free energy and other sequences which interact with specific protein(s) from the chloroplast replication fraction. Apparently the minimal ori sequences reported here contain all the necessary elements for support of chloroplast DNA replication in vitro.

Chloroplast DNA Replication : Mechanism, Enzymes and Replication Origins

Chloroplasts contain circular DNA molecules which are found in low copy number in proplastids but are amplified to very high copy number in actively dividing leaf cells. A double displacement loop (D-loop) mechanism for chloroplast DNA (ctDNA) replication has been proposed, and pairs of replication origins which fit this model have been identified in some species. It appears that ctDNA replication is under the control of at least some nuclear gene products, as genes for DNA polymerase, topoisomerases, DNA primase and other accessory replication proteins have not been reported in the sequenced chloroplast genomes, and ctDNA replication remains active in the absence of active chloroplast transcription or translation. Only a few chloroplast replication proteins have been isolated, and to date most have not been characterized in detail. The mechanism by which ctDNA copy number is regulated during plant development is not known. In this review we summarize the current understanding of ctDNA replication.

Characterization of chloroplast division using the Arabidopsis mutant arc5.

arc5 is a chloroplast division mutant of Arabidopsis thaliana. To identify the role of ARC5 in the chloroplast replication process we have followed the changes in arc5 chloroplasts during their perturbed division. ARC5 does not affect proplastid division but functions at a later stage in chloroplast development. Chloroplasts in developing mesophyll cells of arc5 leaves do not increase in number and all of the chloroplasts in mature leaf cells show a central constriction. Young arc5 chloroplasts are capable of initiating the division process but fail to complete daughter-plastid separation. Wild-type plastids increase in number to a mean of 121 after completing the division process, but in the mutant arc5 the approximately 13 plastids per cell are still centrally constricted but much enlarged. As the arc5 chloroplasts expand and elongate without dividing, the internal thylakoid membrane structure becomes flexed into an undulating ribbon. We conclude that the ARC5 gene is necessary for the completion of the last stage of chloroplast division when the narrow isthmus breaks, causing the separation of the daughter plastids.

Effect of Benzyladenine on Diurnal Changes in DNA Content per Chloroplast and Chloroplast Replication in Intact Bean Leaves

Effect of Benzyladenine on Diurnal Changes in DNA Content per Chloroplast and Chloroplast Replication in Intact Bean Leaves

Effets de l'irradiation γ directe de plantules de tabac sur leur morphologie générale et sur les chloroplastes et la teneur nucléaire en ADN de leur tissu chlorenchymateux

Plantlets of Nicotiana tabacum var. xanthi and of its Dulieu chlorophyll mutant showed a reduced growth and became yellowish when treated with different γ-ray doses (100–1000 Gy). In bom varieties, the size of the cotyledons and of the first leaves was increased and their aspect was modified after irradiation at 500 and, especially, 100 Gy. Furthermore, in the cotyledon green tissue where chloroplast replication is inhibited by irradiation, a marked dose-dependent increase in chloroplast size and starch storage was observed: in the Dulieu variety, at 500 and 1000 Gy, the chloroplast mean area was respectively 74 and 121 times larger than the control area, whereas the starch granule mean area was respectively 28 and 39 times superior to that of the control. Direct irradition of plantlets induced a nucleus endopolyploidization process directly related to the nucleus size in the green tissue of the cotyledons and first leaves of these two tobacco varieties; the endopolyploidization level was very high at 100 Gy (8C, 16C, and > 16C), high at 500 Gy (8C → 16C), and lower but still significant at 1000 Gy (8C). In this tissue, the G2 → M transition point was more sensitive to strong γ irradiation (100–1000 Gy) than the G1 → S transition point. In conclusion, the plantlets of the two tobacco varieties did not show any significant differences in response to strong γ irradiation. Key words: plant morphology, γ irradiation, chloroplasts, nuclear DNA, cytophotometry.

Cell size and chloroplast size in relation to chloroplast replication in light-grown wheat leaves

As part of an investigation into the control of chloroplast replication the number and size of chloroplasts in mesophyll cells was examined in relation to the size of the cells. In first leaves of Triticum aestivum L. and T. monococcum L. the number of chloroplasts in fully expanded mesophyll cells is positively correlated with the plan area of the cells. The linear relationship between chloroplast number per cell and cell plan area is also consistent over a fivefold range of cell size in isogenic diploid and tetraploid T. monococcum. In T. aestivum the chloroplast number per unit cell plan area varies among cells in relation to the size of the chloroplasts. Those cells containing chloroplasts with a relatively small face area have a correspondingly higher density of chloroplasts, and consequently, the total chloroplast area per unit cell plan area is very similar in all the cells. The results indicate that the proportion of the cell surface area covered by chloroplasts is precisely regulated, and that this is achieved during cell development by growth and replication of the chloroplasts.

Stochastic partitioning of chloroplasts at cell division in the alga Olisthodiscus, and compensating control of chloroplast replication

We asked how chloroplasts in a unicellular marine alga are replicated and partitioned at cell division so that each daughter cell will receive the appropriate number of copies. The data were obtained simply by counting chloroplasts in pairs of daughter cells immediately after cell division. The results show that chloroplast partitioning is not always equal; however, it is equal much more often than predicted by the binomial distribution of chloroplast numbers that would be expected if partitioning were strictly random. The parental chloroplasts were partitioned equally in approximately 76% of the divisions, while in the remaining 24% the deviations from equality were very small. To maintain a reasonable range of chloroplast numbers in the face of unequal partitioning, there must be some form of compensating control of chloroplast replication. Our data suggest that daughter cells that receive very large numbers of chloroplasts go directly to the next division without replicating their chloroplasts, while cells with very small numbers of chloroplasts go through two rounds of chloroplast replication before dividing.

Inhibition by Sethoxydim of Chloroplast Biogenesis, Development and Replication in Barley Seedlings

Abstract Chloroplast Biogenesis, Inhibition of Chloroplast Replication, Sethoxydim. Sun-Type Chloroplast Sethoxydim not only blocks leaf growth and development of barley seedlings but also inhibits chloroplast biogenesis at all stages of development from proplastids to prochloroplasts, young and mature chloroplasts. Not only thylakoid synthesis, thylakoid multiplication and grana for­mation are affected, but also chloroplast replication. The chloroplasts of secondary leaves which before the sethoxydim application are in the stage of young, developing chloroplasts, remain in this differentiation stage when treated with sethoxydim. With their ultrastructural characteristics (e.g. lower stacking degree, higher proportion of exposed membranes, a lower thylakoid frequency etc.) they resemble sun-type chloroplasts. In the shoot meristem sethoxydim-treated plants contain only proplastids, whereas the plastids in the shoot meristem of control plants are already in the developmental stage between prochloroplasts to young chloroplasts. Mesophyll cells of sethoxydim-treated plants contain only one third of the chloroplasts found in the controls.

EFFECTS OF CYCLOHEXIMIDE AND CHLORAMPHENICOL ON CHLOROPLAST REPLICATION IN SYNCHRONOUSLY DIVIDING CULTURED CELLS OF EUGLENA GRACILIS

SummaryChloroplast replication and division of Euglena gracilis Z cells cultured synchronoulsy under a diurnal light–dark regime (14:10h light: dark) proceeded in the dark period and were completely inhibited by 5 μg ml1 cycloheximide (CHI) added during the dark period. In the presence of 2 μg ml−1 CHI, chloroplast replication slowed but could be accomplished by the end of a prolonged 24 h dark period, while both the cell and nuclear division were completely inhibited. Chloroplast replication in the presence of 2 μg ml1 CHI was completely inhibited if the dark period was replaced by a light period. Chloramphenicol (CAP) (1 to 2 mg ml−1) slightly inhibited chloroplast replication while accumulation of chlorophyll and total cellular protein were considerably reduced. From the results presented it is concluded that chloroplast replication in synchronously cultured cells of Euglena gracilis is dependent on cytoplasmic protein synthesis that takes place during the dark period rather than chloroplastic protein synthesis, and that a putative protein(s) involved in chloroplast replication is inactivated by light.