Chloroplast nucleoids are highly dynamic in ploidy,number, and structure during angiosperm leaf development

Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post‐meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI‐based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring‐shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20–750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20‐fold even within individual organelles, with average values between 2.6‐fold and 6.7‐fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70–130 copies in chloroplasts of about 7 μm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600–3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed‐field electrophoresis, restriction of high‐molecular‐weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single‐stranded and double‐stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.     

Chloroplast DNA levels and the control of chloroplast division in light-grown wheat leaves

Plastids at different stages of development were isolated from light-grown wheat (Triticum aestivum, var. Maris Dove) seedling leaves, and the average chloroplast DNA (cpDNA) per plastid at each developmental stage was measured directly. In the earliest stages of development, the number of plastids per cell and the amount of cpDNA per cell increased with cell age, but cpDNA per plastid remained constant at between 800 and 1,000 genome copies per plastid. After this phase, plastids per cell continued to increase, but cpDNA per plastid decreased. Subsequently, both plastids per cell and cpDNA per plastid remained constant as cell age increased, the final DNA content being approximately 300 genome copies per plastid. These results are related to previous reports of cpDNA changes during the development of dicotyledonous plants, and to theories about the regulation of chloroplast numbers per cell.     

Localization of plastid DNA replication on a nucleoid structure

Plastids contain multiple copies of the plastid genome that are arranged into discrete aggregates, termed nucleoids. Nucleoid molecular organization and its possible role in ensuring genome continuity have not yet been carefully explored. We examined the relationship between plastid DNA synthesis and nucleoid cytology in the unicellular chrysophyte Ochromonas danica, which is useful for such work because the genomes in each plastid are arranged in a single ring-shaped nucleoid. Immunocytochemical detection of thymidine analog incorporation into replicating DNA revealed that plastid DNA synthesis occurs at several sites along the ring nucleoid simultaneously, and that all plastids of a single cell display similar replication patterns. Plastid DNA replication was observed in G1, S, and G2 phase cells. Pulse-chase-pulse labelling with two different thymidine analogs revealed that new sites are activated as cells progress through the cell cycle while some old sites continue. The double labelling patterns suggest that the individual genomes are arranged consecutively, either singly or in clusters, along the nucleoid perimeter and that the selection of which genome replicates when is a matter of chance. These observations eliminate a number of alternative hypotheses concerning plastid DNA organization, and suggest how cells might maintain a constancy of plastid DNA amount and why plastid genome variants segregate so rapidly during mitosis.     

Identification of the Mitochondrial Genome in the Chrysophyte Alga Ochromonas danica

ABSTRACT. Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (?120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.     

Ins and outs of plastid genome evolution

Recent findings have established cracks in the straight-laced image of the plastid genome as a molecule whose sole function is photosynthesis and whose gene content is highly conserved. Genes for numerous non-photosynthetic functions have been identified. Algal plastid genomes contain many genes with no homologs in angiosperms, and the recent transfer of genes from the plastid to the nuclear genome has been described. Wholesale abandonment of genes encoding photosynthetic and gene-expression functions has occurred in the plastid genomes of a non-green plant and alga. The origins of plastid DNA, its use in phylogenetic studies, and the origins of plastid introns are also reviewed.     

Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle

Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (≤1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10⁻² pg DNA for plastids of 1→8 μm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.     

SecA is plastid-encoded in a red alga: implications for the evolution of plastid genomes and the thylakoid protein import apparatus

Summary Partial sequence analysis of the plastid DNA (ptDNA) from a red alga, Antithamnion sp., revealed the presence of a homologue to the Escherichia coli SecA gene as well as two open reading frames (ORF 510, ORF 179). In addition a sec Y homologue has been detected on the plastid genome by heterologous hybridization. None of these genes has been found in completely sequenced chlorophytic plastid genomes. SecA and secY gene copies were also detected in the ptDNA of a chromophytic alga, indicating that secAY may be ubiquitous in rhodophytes and chromophytes. The significance of these findings for the evolution of plastid genomes and the thylakoid protein import mechanism is discussed.     

Chloroplast DNA content in Dinophysis (Dinophyceae) from different cell cycle stages is consistent with kleptoplasty

Kleptoplasty is the retention of plastids obtained from ingested algal prey, which can remain temporarily functional and be used for photosynthesis by the predator. With a new approach based on cell cycle analysis, we have addressed the question of whether the toxic, bloom-forming dinoflagellate Dinophysis norvegica practice kleptoplasty or if they replicate their own plastid DNA. Dividing (G2) and non-dividing (G1) D. norvegica cells from a natural population were physically separated with a flow cytometer based on their DNA content. Average numbers of nuclear and plastid rDNA copies were quantified with real-time PCR both in the G1 and G2 group. Cells from the G1 group contained 5800 ± 340 copies of nuclear rDNA and 1300 ± 200 copies of plastid rDNA; cells from the G2 group contained 9700 ± 58 copies of nuclear rDNA and 1400 ± 220 copies of plastid rDNA (mean ± SD, n  = 3). The ratio G2/G1 in average rDNA copies per cell was 1.67 for nuclear DNA and 1.07 for plastid DNA. These ratios show that plastid acquisition in D. norvegica is either uncoupled with the cell cycle, or plastids accumulate rapidly in the beginning of the cell cycle owing to feeding, as would be expected in a protist with kleptoplastic behaviour but not in a protist with own plastid replication. In addition, flow cytometry measurements on cells from the same population used for real-time PCR showed that when kept without plastidic prey, live Dinophysis cells lost on average 36% of their plastid phycoerythrin fluorescence in 24 h. Together these findings strongly suggest that D. norvegica does not possess the ability for plastid replication.     

Kinetic Complexity, Homogeneity, and Copy Number of Chloroplast DNA from the Marine Alga Olisthodiscus luteus

The kinetic complexity of chloroplast DNA isolated from the chromophytic alga Olisthodiscus luteus has been determined. Using optical reassociation techniques, it was shown that the plastid DNA of this alga reacted as a single component with a second order rate constant of 4.1 molar⁻¹ and second⁻¹ (Cot_½ 0.24 molar second) under conditions equivalent to 180 millimolar Na⁺ and 60°C. Given the 92 × 10⁵ dalton complexity calculated for this chloroplast genome, an Olisthodiscus cell contains 650 plastome copies. Although this complement remains constant throughout the growth cycle of the organism, the ploidy level of an individual chloroplast shows significant plasticity and is dependent upon the number of chloroplasts present per cell. Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA. Olisthodiscus chloroplast DNA showed no sequence mismatch when thermal denaturation profiles of reassociated chloroplast DNA were examined, thus all plastome copies are essentially identical. Finally, reassociation studies demonstrated that no foldback (short inverted repeat) sequences were present in the plastid genome although significant hairpin loop structures were observed in control nuclear DNA samples.     

Photosynthetic properties of two different soybean suspension cultures

The photosynthetic properties of two commonly used suspension cultured lines, embryogenic and photoautotrophic (PA, SB-1 line) cells of soybean [Glycine max (L.) Merr.] were characterized. We found that compared to the dark green PA cells, the light green embryogenic cells contained fewer and smaller plastids with less-developed thylakoid membranes. The embryogenic cells also contained much lower contents of both chlorophyll and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) protein, an undetectable level of Rubisco small subunit protein, and a very low rate of photosynthesis. While the DNA contents of the nuclear genomes were similar in these two types of cultured cells, the embryogenic cells possessed a markedly lower content of plastid DNA. The 18-year-old PA suspension culture, SB-1, continues to evolve with higher Rubisco and plastid DNA contents than leaves, and with small decreases in nuclear DNA content that appears to mimic changes in chromosome numbers. These findings may prove useful in the application of plastid transformation, particularly when non-leaf or non-green tissues must be used as targets for transformation and plant regeneration.     

Multiple pathways for Cre/lox-mediated recombination in plastids

Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to approximately 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker-free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre-mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans-splicing events in higher plant plastids.     

The complete chloroplast genome of the chlorarachniophyte Bigelowiella natans: evidence for independent origins of chlorarachniophyte and euglenid secondary endosymbionts

Chlorarachniophytes are amoeboflagellate cercozoans that acquired a plastid by secondary endosymbiosis. Chlorarachniophytes are the last major group of algae for which there is no completely sequenced plastid genome. Here we describe the 69.2-kbp chloroplast genome of the model chlorarachniophyte Bigelowiella natans. The genome is highly reduced in size compared with plastids of other photosynthetic algae and is closer in size to genomes of several nonphotosynthetic plastids. Unlike nonphotosynthetic plastids, however, the B. natans chloroplast genome has not sustained a massive loss of genes, and it retains nearly all of the functional photosynthesis-related genes represented in the genomes of other green algae. Instead, the genome is highly compacted and gene dense. The genes are organized with a strong strand bias, and several unusual rearrangements and inversions also characterize the genome; notably, an inversion in the small-subunit rRNA gene, a translocation of 3 genes in the major ribosomal protein operon, and the fragmentation of the cluster encoding the large photosystem proteins PsaA and PsaB. The chloroplast endosymbiont is known to be a green alga, but its evolutionary origin and relationship to other primary and secondary green plastids has been much debated. A recent hypothesis proposes that the endosymbionts of chlorarachniophytes and euglenids share a common origin (the Cabozoa hypothesis). We inferred phylogenies using individual and concatenated gene sequences for all genes in the genome. Concatenated gene phylogenies show a relationship between the B. natans plastid and the ulvophyte-trebouxiophyte-chlorophyte clade of green algae to the exclusion of Euglena. The B. natans plastid is thus not closely related to that of Euglena, which suggests that plastids originated independently in these 2 groups and the Cabozoa hypothesis is false.     

Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

Piecing together the puzzle of parasitic plant plastome evolution

The importance of photosynthesis as a mode of energy production has put plastid genomes of plants under a constant purifying selection. This has shaped the characteristic features of plastid genomes across the entire spectrum of photosynthetic plants and has led to a highly uniform and conserved plastid genome with respect to structure, size, gene order, intron and editing site positions and coding capacity. Parasitic species that have dropped photosynthesis as the main energy provider share striking deviations from the plastid genome norm: multiple rearrangements within the circular chromosome, pseudogenization and gene deletions, promoter losses, intron losses as well as the extensive loss of mRNA editing competence have been reported. The collective loss of larger sets of functionally related genes like those for the plastid NADH–dehydrogenase complex and concomitant losses of RNA polymerase genes together with their target promoters point to “domino effects” where an initial loss might have triggered others. An example, which will be discussed in more detail, is the concomitant loss of the intron maturase gene matK and all introns that are supposedly subject to MatK-dependent splicing in two Cuscuta species.     

SYMBIOTIC ORIGIN OF A NOVEL ACTIN GENE IN THE CRYPTOPHYTE,PYRENOMONAS HELGOLANDII

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment, PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids with this genome ultimately being lost (e.g., as in heterokonts, haptophytes, euglenophytes) when photosynthesis comes under full control of the “host” nucleus. For this to happen, all genes for plastid function must be transferred from the nucleomorph to the nucleus. In this regard, it is generally assumed that nucleomorph genes with functions unrelated to plastid or PC maintenance are lost. Surprisingly, we show here the existence of a novel type of actin gene in the host nucleus of the cryptophyte, Pyrenomonas helgolandii, that has originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage that are unrelated to plastid function. These genes are akin to the products of gene duplication and provide a source of evolutionary novelty that could significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.     

Conservation and Structural Divergence of Organellar DNA and Gene Expression in Non-Photosynthetic Plastids During Ontogenetic Differentiation and Phylogenetic Adaptation

Plastids of non-photosynthetic cells or tissues, such as chromoplasts or leukoplasts, which develop during the course of ontogenetic differentiation contain DNA which is identical to chloroplast DNA with respect to size, organization and gene content. Also in ribosome-deficient bleached plastids, produced in leaves by experimental treatments or mutation, chloroplast DNA remains unaltered. The chloroplast DNA of various bleached mutant strains of Euglena has suffered major deletions or rearrangements, but is, however, never totally lost. Also leukoplasts of parasitic higher plants contain DNA. In the organellar DNA of several parasitic plants photosynthetic genes are conserved. In the heterotrophic flagellate Astasia and in the holoparasite Epifagus virginiana (Orobanchaceae) the size of the plastid DNA is greatly reduced by major deletions and most or all photosynthetic genes or genes related to the chloroplastic respiratory chain are lost. The residual plastid genomes have, however, retained genes for RNAs, tRNAs and ribosomal polypeptides and these are transcribed, although plastidic RNA-polymerase genes are lost in Epifagus. These findings demand the existence of a nuclear-encoded RNA-polymerase. The relevance of the conservation of plastid DNA and of plastidic gene expression in non-photosynthetic cells is discussed, remains, however, at present elusive. Open reading frames of unknown function might be of particular significance for non-photosynthetic plastids.     

The complete plastid genome sequence of the parasitic green alga Helicosporidium sp. is highly reduced and structured

Background  

Loss of photosynthesis has occurred independently in several plant and algal lineages, and represents a major metabolic shift with potential consequences for the content and structure of plastid genomes. To investigate such changes, we sequenced the complete plastid genome of the parasitic, non-photosynthetic green alga, Helicosporidium.