Chloroplast Nucleoids Research Articles

The YlmG protein has a conserved function related to the distribution of nucleoids in chloroplasts and cyanobacteria

BackgroundReminiscent of their free-living cyanobacterial ancestor, chloroplasts proliferate by division coupled with the partition of nucleoids (DNA-protein complexes). Division of the chloroplast envelope membrane is performed by constriction of the ring structures at the division site. During division, nucleoids also change their shape and are distributed essentially equally to the daughter chloroplasts. Although several components of the envelope division machinery have been identified and characterized, little is known about the molecular components/mechanisms underlying the change of the nucleoid structure.ResultsIn order to identify new factors that are involved in the chloroplast division, we isolated Arabidopsis thaliana chloroplast division mutants from a pool of random cDNA-overexpressed lines. We found that the overexpression of a previously uncharacterized gene (AtYLMG1-1) of cyanobacterial origin results in the formation of an irregular network of chloroplast nucleoids, along with a defect in chloroplast division. In contrast, knockdown of AtYLMG1-1 resulted in a concentration of the nucleoids into a few large structures, but did not affect chloroplast division. Immunofluorescence microscopy showed that AtYLMG1-1 localizes in small puncta on thylakoid membranes, to which a subset of nucleoids colocalize. In addition, in the cyanobacterium Synechococcus elongates, overexpression and deletion of ylmG also displayed defects in nucleoid structure and cell division.ConclusionsThese results suggest that the proper distribution of nucleoids requires the YlmG protein, and the mechanism is conserved between cyanobacteria and chloroplasts. Given that ylmG exists in a cell division gene cluster downstream of ftsZ in gram-positive bacteria and that ylmG overexpression impaired the chloroplast division, the nucleoid partitioning by YlmG might be related to chloroplast and cyanobacterial division processes.

The Chlamydomonas Chloroplast HLP Protein Is Required for Nucleoid Organization and Genome Maintenance

The chloroplasts genome (plastome) occurs at high copy numbers per cell. Several chloroplast genome copies are densely packed into nucleoprotein particles called nucleoids. How genome packaging occurs and which proteins organize chloroplast nucleoids are largely unknown. Here, we have analyzed the Chlamydomonas reinhardtii homolog of the bacterial architectural DNA-binding protein HU, the histone-like protein HLP. We show that the Chlamydomonas HLP protein is targeted to chloroplasts and associates with nucleoids. Knockdown of HLP gene expression by RNA interference (RNAi) alters the structure of chloroplast nucleoids and appears to reduce the level of compaction of chloroplast DNA. Unexpectedly, also chloroplast genome copy numbers are significantly decreased in the RNAi strains, suggesting that, in addition to its architectural role in nucleoid formation, the HLP protein is also involved in chloroplast genome maintenance.

A Heterocomplex of Iron Superoxide Dismutases Defends Chloroplast Nucleoids against Oxidative Stress and Is Essential for Chloroplast Development inArabidopsis

There are three iron superoxide dismutases in Arabidopsis thaliana: FE SUPEROXIDE DISMUTASE1 (FSD1), FSD2, and FSD3. Their biological roles in chloroplast development are unknown. Here, we show that FSD2 and FSD3 play essential roles in early chloroplast development, whereas FSD1, which is found in the cytoplasm, does not. An fsd2-1 fsd3-1 double mutant had a severe albino phenotype on agar plates, whereas fsd2 and fsd3 single knockout mutants had pale green phenotypes. Chloroplast development was arrested in young seedlings of the double mutant. The mutant plants were highly sensitive to oxidative stress and developed increased levels of reactive oxygen species (ROS) during extended darkness. The FSD2 and FSD3 proteins formed a heteromeric protein complex in the chloroplast nucleoids. Furthermore, transgenic Arabidopsis plants overexpressing both the FSD2 and FSD3 genes showed greater tolerance to oxidative stress induced by methyl viologen than did the wild type or single FSD2- or FSD3-overexpressing lines. We propose that heteromeric FSD2 and FSD3 act as ROS scavengers in the maintenance of early chloroplast development by protecting the chloroplast nucleoids from ROS.

A member of the Whirly family is a multifunctional RNA- and DNA-binding protein that is essential for chloroplast biogenesis.

‘Whirly’ proteins comprise a plant-specific protein family whose members have been described as DNA-binding proteins that influence nuclear transcription and telomere maintenance, and that associate with nucleoids in chloroplasts and mitochondria. We identified the maize WHY1 ortholog among proteins that coimmunoprecipitate with CRS1, which promotes the splicing of the chloroplast atpF group II intron. ZmWHY1 localizes to the chloroplast stroma and to the thylakoid membrane, to which it is tethered by DNA. Genome-wide coimmunoprecipitation assays showed that ZmWHY1 in chloroplast extract is associated with DNA from throughout the plastid genome and with a subset of plastid RNAs that includes atpF transcripts. Furthermore, ZmWHY1 binds both RNA and DNA in vitro. A severe ZmWhy1 mutant allele conditions albino seedlings lacking plastid ribosomes; these exhibit the altered plastid RNA profile characteristic of ribosome-less plastids. Hypomorphic ZmWhy1 mutants exhibit reduced atpF intron splicing and a reduced content of plastid ribosomes; aberrant 23S rRNA metabolism in these mutants suggests that a defect in the biogenesis of the large ribosomal subunit underlies the ribosome deficiency. However, these mutants contain near normal levels of chloroplast DNA and RNAs, suggesting that ZmWHY1 is not directly required for either DNA replication or for global plastid transcription.

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the MossPhyscomitrella patens

RecA protein is widespread in bacteria, and it plays a crucial role in homologous recombination. We have identified two bacterial-type recA gene homologs (PprecA1, PprecA2) in the cDNA library of the moss Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA2 to the green fluorescent protein (GFP) caused a targeting of PpRecA2 to the chloroplasts. Mutational analysis showed that the first AUG codon acts as initiation codon. Fusion of the full-length PpRecA2 to GFP caused the formation of foci that were colocalized with chloroplast nucleoids. The amounts of PprecA2 mRNA and protein in the cells were increased by treatment with DNA damaging agents. PprecA2 partially complemented the recA mutation in Escherichia coli. These results suggest the involvement of PpRecA2 in the repair of chloroplast DNA.

The nucleocytosolic and chloroplast cycle in the green chlorococcal alga Scenedesmus obliquus (Chlorophyceae, Chlorococcales) grown under various temperatures

V. Cepák, P. Přibyl, M. Vítová and V. Zachleder. 2007. The nucleocytosolic and chloroplast cycle in the green chlorococcal alga Scenedesmus obliquus (Chlorophyceae, Chlorococcales) grown under various temperatures. Phycologia 46: 263–269. DOI: 10.2216/06-39.1Synchronous cultures of the green alga Scenedesmus obliquus were grown at different temperatures (15°C, 20°C, 26°C, and 33°C) and the effect of temperature on cell cycle events as attainment of commitment points, division of cell nuclei and chloroplast nucleoids, and the length of the cell cycle phases were investigated. The length of the cell cycle was prolonged markedly with decreasing temperature (from 12 h at 33°C to 70 h at 15°C), while the number of daughter cells per one mother cell remained unchanged. The size of mother cells and consequently the size of daughter cells increased with decreasing temperature. Within the range of temperatures used, no effect on the cytomorphology of chloroplast nucleoids (shape, size, and spatial distribution) was observed. The nucleoids were of various shapes (spherical, elongated, rodlike), evenly distributed, and divided asynchronously at all temperatures used. They were distributed randomly in the chloroplast during the entire cell cycle. The mean number of nucleoids in daughter cells released at 33°C was about 10. The number of chloroplast nucleoids increased proportionally to cell (chloroplast) size. Thus, the number of nucleoids in daughter cells increased with decreasing temperature. Division of chloroplast nucleoids preceded cell nuclei division at all temperatures used. Temperature affected the nucleocytoplasmic and chloroplast compartments distinctly. With decreasing temperature, the cell size of daughter cells increased, and the length of the cell cycle and its phases was prolonged. The number of mitoses and consequently the number of daughter cells remained, however, unchanged, while the number of nucleoids increased during the cell cycle proportionally to the increase in cell size at any given temperature. No temperature compensation, which could indicate the effect of some endogenous temperature-independent timers, was observed.

DNA binding and partial nucleoid localization of the chloroplast stromal enzyme ferredoxin:sulfite reductase.

Sulfite reductase (SiR) is an important enzyme catalyzing the reduction of sulfite to sulfide during sulfur assimilation in plants. This enzyme is localized in plastids, including chloroplasts, and uses ferredoxin as an electron donor. Ferredoxin-dependent SiR has been found in isolated chloroplast nucleoids, but its localization in vivo or in intact plastids has not been examined. Here, we report the DNA-binding properties of SiRs from pea (PsSiR) and maize (ZmSiR) using an enzymatically active holoenzyme with prosthetic groups. PsSiR binds to both double-stranded and single-stranded DNA without significant sequence specificity. DNA binding did not affect the enzymatic activity of PsSiR, suggesting that ferredoxin and sulfite are accessible to SiR molecules within the nucleoids. Comparison of PsSiR and ZmSiR suggests that ZmSiR does indeed have DNA-binding activity, as was reported previously, but the DNA affinity and DNA-compacting ability are higher in PsSiR than in ZmSiR. The tight compaction of nucleoids by PsSiR led to severe repression of transcription activity in pea nucleoids. Indirect immunofluorescence microscopy showed that the majority of SiR molecules colocalized with nucleoids in pea chloroplasts, whereas no particular localization to nucleoids was detected in maize chloroplasts. These results suggest that SiR plays an essential role in compacting nucleoids in plastids, but that the extent of association of SiR with nucleoids varies among plant species.

Atomic Force Microscopy Dissects the Hierarchy of Genome Architectures in Eukaryote, Prokaryote, and Chloroplast

Because of its applicability to biological specimens (nonconductors), a single-molecule-imaging technique, atomic force microscopy (AFM), has been particularly powerful for visualizing and analyzing complex biological processes. Comparative analyses based on AFM observation revealed that the bacterial nucleoids and human chromatin were constituted by a detergent/salt-resistant 30-40-nm fiber that turned into thicker fibers with beads of 70-80 nm diameter. AFM observations of the 14-kbp plasmid and 110-kbp F plasmid purified from Escherichia coli demonstrated that the 70-80-nm fiber did not contain a eukaryotic nucleosome-like "beads-on-a-string" structure. Chloroplast nucleoid (that lacks bacterial-type nucleoid proteins and eukaryotic histones) also exhibited the 70-80-nm structural units. Interestingly, naked DNA appeared when the nucleoids from E. coli and chloroplast were treated with RNase, whereas only 30-nm chromatin fiber was released from the human nucleus with the same treatment. These observations suggest that the 30-40-nm nucleoid fiber is formed with a help of nucleoid proteins and RNA in E. coli and chroloplast, and that the eukaryotic 30-nm chromatin fiber is formed without RNA. On the other hand, the 70-80-nm beaded structures in both E. coli and human are dependent on RNA.

MORPHOLOGICAL CHANGES IN MITOCHONDRIAL AND CHLOROPLAST NUCLEOIDS AND MITOCHONDRIA DURING THECHLAMYDOMONAS REINHARDTII(CHLOROPHYCEAE) CELL CYCLE1

Morphological changes in the organellar nucleoids and mitochondria of livingChlamydomonas reinhardtiiDang were examined during the cell cycle under conditions of 12:12 light:dark. The nucleoids were stained with SYBR‐Green I, and the mitochondria were stained with 3,3‐dihexyloxacarbocyanine iodide. An mocG33 mutant, which contains one large chloroplast nucleoid throughout the cell cycle, was used to distinguish between the mitochondrial and chloroplast nucleoids. Changes in the total levels of organellar DNA levels were assessed by real‐time PCR. Each of the G1, S, M, and Smt,cp phases was estimated. At the start of the light period, the new daughter cells were in G1and contained about 30 mitochondrial and 10 chloroplast nucleoids, which were dispersed and had diameters of 0.1 and 0.2 μm, respectively. During the G1phase of the light period, and at the start of the S phase, both nucleoids formed short thread‐like or bead‐like structures, probably divided, and increased continuously in number, concomitantly with DNA synthesis. The nucleoids probably became smaller due to the decrease in DNA of each particle and were indistinguishable. The cells in the S and M phases contained extremely high numbers of scattered nucleoids. However, in the G1phase of the dark period, the nucleoids again formed short thread‐like or bead‐like structures, probably fused, and decreased in number. The mitochondria appeared as tangled sinuous structures that extended throughout the cytoplasm and resembled a single large mitochondrion. During the cell cycle, the numbers of mitochondrial nucleoids and sinuous structures varied relative to one another.

Visualization of DNA-containing structures in various species ofChlorophyta, Rhodophyta andCyanophyta using SYBR green I dye

We developed an alternative method of staining cell nuclei and chloroplast nucleoids of algal cells using SYBR Green I (the fluorescent dye used commonly for detecting dsDNA in agarose and polyacrylamide gels as an alternative to highly mutagenic ethidium bromide and for DNA staining of viruses and bacteria followed by flow cytometry, digital image analysis or real-time PCR), which enabled routine staining in vivo. Cells do not need to be fixed or treated chemically or physically before staining, thus the shape, size and position of DNA-containing structures are not affected. The fluorescence signal is sharp and reproducible. Examples of application of the method are shown in color microphotographs for representatives of eukaryotic algae from the taxa Chlorophyta, Rhodophyta and the prokaryotic Cyanophyta. The method is also useful for studying progress of the cell cycle in algal cells dividing by multiple fission, as shown by observation of changes in nuclear number during the cell cycle of the green alga Chlamydomonas reinhardtii and Scenedesmus quadricauda. Staining with SYBR Green I can be recommended as a fast, safe and efficient method for the detection of DNA-containing structures in vivo.

Visualization of plastid nucleoids in situ using the PEND-GFP fusion protein.

Plastid DNA is a circular molecule of 120-150 kbp, which is organized into a protein-DNA complex called a nucleoid. Although various plastids other than chloroplasts exist, such as etioplasts, amyloplasts and chromoplasts, it is not easy to observe plastid nucleoids within the cells of many non-green tissues. The PEND (plastid envelope DNA-binding) protein is a DNA-binding protein in the inner envelope membrane of developing chloroplasts, and a DNA-binding domain called cbZIP is present at its N-terminus. We made various PEND-green fluorescent protein (GFP) fusion proteins using the cbZIP domains from various plants, and found that they were localized in the chloroplast nucleoids in transient expression in leaf protoplasts. In stable transformants of Arabidopsis thaliana, PEND-GFP fusion proteins were also localized in the nucleoids of various plastids. We have succeeded in visualizing plastid nucleoids in various intact tissues using this stable transformant. This technique is useful in root, flower and pollen, in which it had been difficult to observe plastid nucleoids. The relative arrangement of nucleoids within a chloroplast was kept unchanged when the chloroplast moved within a cell. During the division of plastid, nucleoids formed a network structure, which made possible equal partition of nucleoids.

DNA gyrase is involved in chloroplast nucleoid partitioning.

DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 4',6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology.

The DNA-binding protease, CND41, and the degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase in senescent leaves of tobacco.

Plastids bear their own genome, organized into DNA-protein complexes (nucleoids). Recently, we identified a DNA-binding protease (CND41) in the chloroplast nucleoids of cultured tobacco (Nicotiana tabacum L.) cells. In this study, we examine the biochemical function of this novel DNA-binding protease, particularly in senescent leaves, because antisense tobacco with a reduced amount of CND41 showed retarded senescence. Nitrogen-depletion experiments clearly showed that CND41 antisense tobacco maintained green leaves and constant protein levels, especially ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), throughout the whole plant, whereas wild-type tobacco showed marked senescence and the reduction of protein levels in the lower leaves. In vitro analyses confirmed that CND41 showed proteolytic activity at physiological pH when denatured Rubisco was used as the substrate. These results suggest that CND41 is involved in Rubisco degradation and the translocation of nitrogen during senescence. The possible regulation of protease activity of CND41 through DNA-binding is discussed.

The effect of nalidixic acid on growth and reproductive events in nucleocytosolic and chloroplast compartments in the algaScenedesmus quadricauda

The courses of rRNA accumulation, DNA replication, and nuclear division were followed both in the chloroplast and the nucleocytosolic compartments during the cell cycle in synchronized populations of the chlorococcal alga Scenedesmus quadricauda. Control and nalidixic acid-treated cultures were compared. Nalidixic acid (150 mg/L) was added either at the beginning of the cell cycle or consecutively during the cell cycle to subcultures transferred into the dark. If the inhibitor was applied at the beginning of the cell cycle, chloroplast DNA did not replicate and nucleoids did not divide. Chloroplast division, however, was coordinated in a timely fashion with cytokinesis even under conditions of blocked chloroplast DNA replication. While the growth rate was slowed down, the courses of reproductive processes in the nucleocytosolic compartment were not affected and their timing and the number of rounds were coordinated with growth rate as in the control culture. The rate of cytosolic rRNA synthesis was lower but no apparent effect was seen on the amount of rRNA that accumulated during the cell cycle. In contrast, lower levels of chloroplast rRNA were found at the end of the cell cycle compared with the control culture. Experiments in which cells were transferred to the dark during the cell cycle showed that the inhibitor affected none of the reproductive events in the nucleocytosolic compartment. In the chloroplast compartment, DNA replication was inhibited in inhibitor-treated cultures, but was unaffected in controls. The chloroplast nucleoids themselves divided even in the presence of the inhibitor, reducing their DNA content to a level which corresponded to that in freshly formed control daughter cells.

Isolation and Partial Characterization of DNA Topoisomerase I from the Nucleoids of White Mustard Chloroplasts

DNA topoisomerase was isolated for the first time from nucleoids of white mustard (Sinapis alba L.) chloroplasts. The enzyme had a molecular weight of 70 kDa; it was ATP-independent, required the presence of mono- (K+) and bivalent (Mg2+) cations, and was capable of relaxing both negatively and positively supercoiled DNA. These results suggest that the enzyme isolated belongs to type IB DNA topoisomerases.

MFP1 is a thylakoid-associated, nucleoid-binding protein with a coiled-coil structure.

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein-DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.

CND41, a chloroplast nucleoid protein that regulates plastid development, causes reduced gibberellin content and dwarfism in tobacco

CND41, a DNA binding protein of chloroplast nucleoids, may function as a negative regulator of chloroplast gene expression. The reduction of CND41 in an antisense transformant accelerated plastid development in shoot apex cells and early young leaves, and caused a dwarf phenotype and altered leaf morphology. Plant height and leaf shape could be restored almost to those of the wild type by application of gibberellins (GAs), clearly indicating that a reduction in GA content was a prime cause of the dwarf phenotype in CND41 antisense transformants. The transformants had reduced endogenous levels of active gibberellin (GA1), a biologically active GA, compared to those of wild‐type plants. Possible relationships between chloroplast development affected by CND41 function and GA biosynthesis are discussed.

Reversible DNA Compaction by Sulfite Reductase Regulates Transcriptional Activity of Chloroplast Nucleoids

The transcriptional activity of nucleoids changes during plastid development, presumably due to the morphological and molecular differences of the nucleoids. Pea chloroplast nucleoids have an abundant 70-kDa protein identified as sulfite reductase (SiR) that can compact DNA. Using an in vitro transcription assay, we show here that heparin increased the transcriptional activity of chloroplast nucleoids with concomitant release of SiR. Using a fluorometric method we developed for analyzing DNA compaction, we found that the fluorescence intensity of chloroplast DNA stained with 4',6-diamidino-2-phenylindole was decreased by the addition of SiR and increased by the subsequent addition of heparin. Addition of exogenous SiR increased the compaction of isolated nucleoids, and the addition of heparin relaxed it. SiR effectively repressed the in vitro transcription activity of nucleoids and counteracted the activation by heparin. These results suggest that SiR regulates the transcriptional activity of chloroplast nucleoids through changes in DNA compaction.

The DNA-compacting protein DCP68 from soybean chloroplasts is ferredoxin:sulfite reductase and co-localizes with the organellar nucleoid.

The multiple copies of the chloroplast genome (plastome) are condensed and organized into nucleoids by a set of proteins. One of these, the DNA-binding protein DCP68 from soybean, has previously been shown to compact DNA and to inhibit DNA synthesis in vitro. N-terminal amino acid analysis and the absorption spectrum of the purified protein suggest that DCP68 is the siroheme protein sulfite reductase, a ferredoxin-dependent enzyme that participates in sulfur assimilation for cysteine and methionine biosynthesis. The in vivo association of this protein with chloroplast nucleoids was confirmed by immuno-colocalization with antibodies against sulfite reductase from Arabidopsis thaliana. These results suggest that DCP68 is a bifunctional chloroplast protein that participates in reductive sulfur assimilation and plays a role in organellar nucleoid organization. The fact that dephosphorylation by alkaline phosphatase affects the binding of purified DCP68 to DNA in vitro might be indicative of the way the interaction of the protein with the nucleoid is regulated in vivo.

Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia.

Differentiation into a tracheary element (TE) is a typical example of programmed cell death (PCD) in the developmental processes of vascular plants. In the PCD process the TE degrades its cellular contents and becomes a hollow corpse that serves as a water conduct. Using a zinnia (Zinnia elegans) cell culture we obtained serial observations of single living cells undergoing TE PCD by confocal laser scanning microscopy. Vital staining was performed and the relative fluorescence intensity was measured, revealing that the tonoplast of the swollen vacuole in TEs loses selective permeability of fluorescein just before its physical rupture. After the vacuole ruptured the nucleus was degraded rapidly within 10 to 20 min. No prominent chromatin condensation or nuclear fragmentation occurred in this process. Nucleoids in chloroplasts were also degraded in a similar time course to that of the nucleus. Degradations did not occur in non-TEs forced to rupture the vacuole by probenecid treatment. These results demonstrate that TE differentiation involves a unique type of PCD in which active and rapid nuclear degradation is triggered by vacuole rupture.