DIVERSITY OF PLASTID DNA CONFIGURATION AMONG CLASSES OF EUKARYOTE ALGAE1

Information is presented concerning the overall arrangement of plastid DNA (ptDNA) in plastids of approximately 100 spp. of eukaryote algae, representing all classes. The three-dimensional arrangement of the ptDNA was assessed by study of both living and fixed material, stained with the DNA fluorochrome 4′,6-diamidino-2-phenylindole (DAPI), using both phase and fluorescence microscopy. The widespread occurrence of two major types of ptDNA configuration known from prior electron microscopy studies was confirmed. These are (1) DNA densities (nucleoids) of variable size and morphology, scattered throughout the plastid, and (2) a ring nucleoid, beaded or unbeaded, lying just within the girdle lamella. Type 1 is characteristic of Rhodophyta, Dinophyta, Chlorophyta, Cryptophyta, Prymnesiophyceae and Eustigmatophyceae (with one exception). Type 2 is characteristic of Phaeophyceae, Bacillariophyceae, Raphidophyceae, Chrysophyceae (except silicoflagellates and organisms such as Synura and Dinobryon), and Xanthophyceae (with the exception of Vaucheria and three genera known to lack girdle lamellae, Bumilleria, Bumilleriopsis, and Pseudobumilleriopsis). Some of these exceptional forms, as well as Euglenophyta, have configurations of ptDNA not previously recognized. In all the configurations observed, the DNA of a single plastid could be interpreted as being in continuity. This character of plastids appears to be stable under varied conditions of growth and at differing stages of the life cycle, where examined, and has confirmed the reclassification made on other grounds of several taxonomic entities. It has also revealed new questionable classifications. Since DAPI staining is far simpler than serial sectioning for electron microscopy in revealing ptDNA architecture, use of the technique may be valuable for future studies of numerous organisms, both to help in their identification and as an aid to unravelling major taxonomic affinities. In light of the endosymbiont hypothesis, plastid characters may require as great attention as those of the remainder of the cell.     

STRUCTURAL ORGANIZATION OF PLASTID DNA IN TWO ANOMALOUSLY PIGMENTED DINOFLAGELLATES1

The structural organization of DNA in the plastids of two anomalously pigmented dinoflagellates, Glenodinium foliaceum Stein and Gyrodinium aureolum Hulburt, was determined using the DNA-specific fluorochrome DAPI and correlated with TEM observations. The plastids of G. foliaceum were found to possess both a peripheral DNA ring and isolated point nucleoids. This arrangement was shown to be similar to that of the diatom Asterionella formosa Hass. and may be characteristic of the Bacillariophyceae. G. aureolum exhibited a novel distribution of plastid. DNA as one or two beaded bands, whereas the plastids of the similarly pigmented haptophyte, Emiliania huxleyi (Lohm.) Hay & Mohler, possessed scattered point nucleoids. These findings support the idea that G. foliaceum harbours an endosymbiotic diatom, but suggest that the plastids of G. aureolum and E. huxleyi are unrelated. The use of plastid DNA configuration as a phylogenetic marker is considered.     

ENDOBACTERIA IN THE DIATOM PINNULARIA (BACILLARIOPHYCEAE). I. “SCATTERED ct‐NUCLEOIDS” EXPLAINED: DAPI–DNA COMPLEXES STEM FROM EXOPLASTIDIAL BACTERIA BORING INTO THE CHLOROPLASTS1,2

A local strain of the pennate diatom Pinnularia cf. “nobilis” was investigated using cytochemistry and fluorescence and EM techniques. The regular perforation of the chloroplasts of P. “nobilis” and the lack of a typical diatom pyrenoid were confirmed at the ultrastructural level. Cavities and channels in the complex secondary plastid were found to harbor symbiotic bacteria, and their DNA elicited DAPI fluorescence. Wheat germ agglutinin, labeling bacteria walls, elicited a similar fluorescence pattern. Previous speculation that the apochlorotic DNA‐positive dots in the plastids of several Pinnularia species are “scattered ct‐nucleoids” is thus refuted. Bacteria were rod shaped and gram negative. They resided in the lumen of the endoplasmic reticulum (ER) during host interphase confined to the specialized ER compartment housing the secondary plastid, that is, to the space between the third and the fourth membrane profile, encircling the chloroplast. TEM images of chemically and cryofixed cells revealed that cavities resulted from the interaction of bacteria with the plastid according to the following sequence, alignment, attachment, deformation, and disintegration. This occurred without visible injury to the primary chloroplast envelope or the relict cell membrane of the reduced ancestral red alga that surrounds the chloroplast. The patterned arrangement of bacteria suggests recognition sites on the vestigial cell membrane, thought to interact with surface groups on the bacteria. The intimate association between bacteria and secondary plastid inside the common specialized ER cisterna suggests they form a functional unit. Comparison of thylakoid profiles, disrupted by bacteria in Pinnularia, with those disrupted by the pyrenoid in other pennate diatoms (e.g. Trachyneis) revealed a significant ultrastructural resemblance. No aposymbiotic Pinnularia cells were found at the sampling site.     

THE LACK OF CHLOROPLAST DNA IN ACETABULARIA MEDITERRANEA (ACETABULUM) (CHLOROPHYCEAE): A REINVESTIGATION1

Three features of chloroplast DNA (cpDNA) in plastids isolated from Acetabularia mediterranea (acetabulum) were analyzed after staining the organelles with the fluorochrome 4′6-diamidino-2-phenyl indole (DAPI): (1) number of chloroplasts exhibiting DNA fluorescence, (2) number of nucleoids per plastid, and (3) nucleoid morphology. In vegetative Acetabularia cells only half of the total chloroplast population comprising several millions displayed the whitish-blue fluorescence of the DNA/DAPI complex. This percentage remained stable independent of whether cells were grown in supplemented natural sea water or enriched synthetic sea water. A single nucleoid, widely differing in size and morphology among the organelles, was characteristic of 76–81% of chloroplasts with DNA. Less than 20% contained two nucleoids, and in rare cases three or four nucleoids were present. The pattern of nucleoid numbers followed a Poisson distribution in one experiment, if calculated with the intrinsic mean of the observed data. In two other experiments, however, a significant difference existed between observed and expected values for a Poisson distribution according to the Chisquared test. After secondary enlargement of portions of the negatives, the nucleoids’substructure was disclosed and found to consist of brightly fluorescent spots interspersed by unstained regions The lack of cpDNA in Acetabularia cells appears to be brought about by (1) the polarized pattern of growth and translation confined to the apical region of the single cell and (2) the cpDNA arrangement in a single nucleoid acentrically located in the organelle. A scheme for the evolution of a chloroplast population having plastids without DNA is proposed. In theory the lack of cpDNA could arise in each plant, since chloroplasts never evolved a mitotic-like spindle to ensure the equal distribution of genetic material. The different nucleoid arrangement in most other plants, however, efficiently counteracts this ‘carelessness of nature’     

Protein translocation into and within cyanelles (Review)

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria in morphology, pigmentation and, especially, in the presence of a peptidoglycan wall situated between the inner and outer envelope membranes. However, it is now clear that cyanelles in fact are primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high plastid gene content of the Porphyra purpurea rhodoplast and the peptidoglycan wall of glaucocystophyte cyanelles. This means that the import apparatus of all primary plastids should be homologous. Indeed, heterologous in vitro import can now be shown in both directions, provided a phenylalanine residue essential for cyanelle import is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved explaining the efficient heterologous import of native precursors from C. paradoxa. With respect to conservative sorting in cyanelles, both the Sec and Tat pathways could be demonstrated. Another cyanobacterial feature, the dual location of the Sec translocase in thylakoid and inner envelope membranes, is also unique to cyanelles. For the first time, protease protection of internalized lumenal proteins could be shown for cyanobacteria-like, phycobilisome-bearing thylakoid membranes after import into isolated cyanelles.     

The Cyanelles of Cyanophora Paradoxa

The cyanelles of Cyanophora paradoxa, plastids surrounded by a peptidoglycan wall, are considered as a surviving example for an early stage of plastid evolution from endosymbiotic cyanobacteria. We highlight the model character of the system by focusing on three aspects: “organelle wall” structure, plastid genome organization, and protein translocation.

The biosynthetic pathway for cyanelle peptidoglycan appears to be analogous to that in Escherichia coli. Also, the basic structure of this peculiar organelle wall corresponds to that of the E. coli sacculus, with one notable exception: the C-1 carboxyl group of the D-isoglutamyl residue is partially amidated with N-acetylputrescine. Cyanelles harbor on their completely sequenced 135.6-kb genome genes for approximately 150 polypeptides, many of which are nucleus encoded in higher plants. Nevertheless, there are striking parallels in genome organization between cyanelles (and other primitive plastids) and higher plant chloroplasts. The transit sequences of nucleus-encoded cyanelle preproteins resemble stroma targeting peptides of higher plant chloroplast precursors. Heterologous import of precursors from C. paradoxa into isolated pea chloroplasts is possible and vice versa. Cyanelles are considered to represent a very early, diverging branch of plastid evolution and are derived from the semiautonomous endosymbiont that had already abandoned about 90% of its genetic information but still retained its prokaryotic wall. Recent data on the molecular biology of cyanelles and rhodoplasts are consistent with the assumption of a primary endosymbiotic event that was not only monophyletic with respect to the cyanobacterial invader, but also singular.

Cyanophora paradoxa is the best-investigated member of the glaucocystophyceae, phototrophic protists containing cyanelles, that is, plastids stabilized by a peptidoglycan-containing envelope. The classification of this group, comprising only eight (mostly monotypic) genera, is also based on parallels in morphology and organization of the “host cells” (Kies, 1992). Recently, this was corroborated by 16S and 18S rRNA-based phylogenetic analysis (Helmchen et al., 1995; Bhattacharya et al, 1995). Apart from C. paradoxa, only Glaucocystis nostochinearum can be grown at a reasonable rate. Thus, biochemical and molecular genetic data are mostly available for C. paradoxa and more precisely for the isolate 555UTEX (Pringsheim) that is kept in the major culture collections of algae. Biochemical work done on C. paradoxa and the sequencing of individual cyanelle genes have been described in several recent reviews (Schenk, 1992; Löffelhardt and Bohnert, 1994a,b). Here we discuss three topics: the cyanelle wall, aspects deduced from the complete cyanelle genome sequence, and protein translocation into and within cyanelles.     

INTERMEDIATE FEATURES OF CYANELLE DIVISION OF CYANOPHORA PARADOXA (GLAUCOCYSTOPHYTA) BETWEEN CYANOBACTERIAL AND PLASTID DIVISION1

Cyanelles of glaucocystophytes may be the most primitive of the known plastids based on their peptidoglycan content and the sequence phylogeny of cyanelle DNA. In this study, EM observations have been made to characterize the cyanelle division of Cyanophora paradoxa Korshikov and to gain insights into the evolution of plastid division. Constriction of cyanelles involves ingrowth of the septum at the cleavage site with the inner envelope membrane invaginating at the leading edge and the outer envelope membrane invaginating behind the septum. This means the inner and outer envelope membranes do not constrict simultaneously as they do in plastid division in other plants. The septum and the cyanelle envelope became stained after a silver‐methenamine staining was applied for in situ detection of polysaccharides. Septum formation was inhibited by β‐lactams and vancomycin, which are potent inhibitors of bacterial peptidoglycan biosynthesis. These results suggest the presence of peptidoglycan at the septum and the cyanelle envelope. In dividing cyanelles, a single electron‐dense ring (cyanelle ring) was observed on the stromal face of the inner envelope membrane at the isthmus, but no ring‐like structures were detected on the outer envelope membrane. Thus a single, stromal cyanelle ring such as this is quite unique and also distinct from FtsZ rings, which are not detectable by TEM. These features suggest that the cyanelle division of glaucocystophytes represents an intermediate stage between cyanobacterial and plastid division. If monophyly of all plastids is true, the cyanelle ring and the homologous inner plastid dividing ring might have evolved earlier than the outer plastid dividing ring.     

OBSERVATIONS ON PLASTID FINE-STRUCTURE IN THE HOLOPARASITIC ANGIOSPERM EPIFAGUS VIRGINIANA

The ultrastructure of plastids in cortex and phloem parenchyma cells of Epifagus virginiana (L.) Bart. is described. Based upon morphology and content, several distinct plastid types appear to exist. “Tubular” complexes, lipid globules and electron dense inclusions in different arrangements appear to account for the degree of plastid variability. When results obtained with Epifagus are compared with those obtained by others for a closely related genus, a striking parallel is shown to exist.     

Analyses of ribosomal RNA sequences from glaucocystophyte cyanelles provide new insights into the evolutionary relationships of plastids

Glaucocystophyte algae (sensu Kies, Berl. Deutsch. Bot. Ges. 92, 1979) contain plastids (cyanelles) that retain the peptidoglycan wall of the putative cyanobacterial endosymbiont; this and other ultrastructural characters (e.g., unstacked thylakoids, phycobilisomes) have suggested that cyanelles are primitive plastids that may represent undeveloped associations between heterotrophic host cells (i.e., glaucocystophytes) and cyanobacteria. To test the monophyly of glaucocystophyte cyanelles and to determine their evolutionary relationship to other plastids, complete 16S ribosomal RNA sequences were determined for Cyanophora paradoxa, Glaucocystis nostochinearum, Glaucosphaera vacuolata, and Gloeochaete wittrockiana. Plastid rRNAs were analyzed with the maximum-likelihood, maximumparsimony, and neighbor joining methods. The phylogenetic analyses show that the cyanelles of C. paradoxa, G. nostochinearum, and G. wittrockiana form a distinct evolutionary lineage; these cyanelles presumably share a monophyletic origin. The rDNA sequence of G. vacuolata was positioned within the nongreen plastid lineage. This result is consistent with analyses of nuclear-encoded rRNAs that identify G. vacuolata as a rhodophyte and support its removal from the Glaucocystophyta. Results of a global search with the maximumlikelihood method suggest that cyanelles are the first divergence among all plastids; this result is consistent with a single loss of the peptidoglycan wall in plastids after the divergence of the cyanelles. User-defined tree analyses with the maximum-likelihood method indicate, however, that the position of the cyanelles is not stable within the rRNA phylogenies. Both maximumparsimony and neighbor-joining analyses showed a close evolutionary relationship between cyanelles and nongreen plastids; these phylogenetic methods were sensitive to inclusion/exclusion of the G. wittrockiana cyanelle sequence. Base compositional bias within the G. wittrockiana 16S rRNA may explain this result. Taken together the phylogenetic analyses are interpreted as supporting a near-simultaneous radiation of cyanelles and green and nongreen plastids; these organelles are all rooted within the cyanobacteria.Correspondence to: D. Bhattacharya     

MORPHOLOGY OF DNA CONTAINING STRUCTURES (NUCLEOIDS) AS A PROSPECTIVE CHARACTER IN CYANOPHYTE TAXONOMY1

DNA containing structures (nucleoids) were visualized by 4′, 6-diamidino-2-phenylindole (DAPI) fluorescent staining in two groups of cyanophytes (59 filamentous oscillatorialean species and 12 coccal Synechococcus-like organisms) to test the possibility of using nucleoid morphology in cyanophyte taxonomy. The morphology of nucleoids (size, shape, and structure) in oscillatorialean species is specific for individual families. The morphology of the nucleoid in Synechococcus-like species agrees from the proposed separation of the genus Cyanothece from Synechococcus. A much different nucleoid morphology in three species of Cyanothece suggests that these species should be separated into a new genus. On the basis of other characters, the species could be returned to the genus Cyanobacterium. My results indicate that the morphology of nucleoids is a valuable character in the classification of the cyanophytes examined; thus, it is a prospective feature that could be used in the taxonomy of other groups of cyanophytes. Additionally, DAPI staining is not a complicated procedure. The new character is easy to see in samples taken from nature, both living and preserved.     

The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining

Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination.The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates (‘nucleoids’) which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote.Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 × as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8–16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.     

The role of chloroplast membranes in the location of chloroplast DNA during the greening ofPhaseolus vulgaris etioplasts

Summary The location of DNA containing nucleoids has been studied in greening bean (Phaseolus vulgaris L.) etioplasts using electron microscopy of thin sections and the staining of whole leaf cells with the fluorochrome DAPI. At 0 hours illumination a diffuse sphere of cpDNA surrounds most of the prolamellar body. It appears to be made up of a number of smaller nucleoids and can be asymmetric in location. The DNA appears to be attached to the outside of the prolamellar body and to prothylakoids on its periphery. With illumination the nucleoid takes on a clear ring-like shape around the prolamellar body. The maximum development of the ring-like nucleoid at 5 hours illumination is associated with the outward expansion of the prolamellar body and the outward growth of the prothylakoids. At 5 hours the electron transparent areas lie in between the prothylakoids radiating out from the prolamellar body. Between 5 hours and 15 hours observations are consistent with the growing thylakoids separating the nucleoids as the prolamellar body disappears and the chloroplast becomes more elongate. At 15 hours the fully differentiated chloroplast has discrete nucleoids distributed throughout the chloroplast with evidence of thylakoid attachment. This is the SN (scattered nucleoid) distribution ofKuroiwa et al. (1981) and is also evident in 24 hours and 48 hours chloroplasts which have more thylakoids per granum. The changes in nucleoid location occur without significant changes in DNA levels per plastid, and there is no evidence of DNA or plastid replication.The observations indicate that cpDNA partitioning in dividing SN-type chloroplasts could be achieved by thylakoid growth and effectively accomplish DNA segregation, contrasting with envelope growth segregating nucleoids in PS-type (peripheral scattered nucleoids) chloroplasts. The influence of plastid development on nucleoid location is discussed.     

Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny

Plastids and mitochondria, the DNA‐containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co‐transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild‐type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS‐92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid‐transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.     

Pattern of the plastid division in spore mother cells of the hornwortAnthoceros punctatus

Relative changes in plastid DNA content in each stage of plastid division were investigated in order to better understand the division cycle of plastids in spore mother cells in the horwortAnthoceros punctatus. Samples of cells stained with DAPI were observed with epifluorescence microscopy and CHIAS. In spore mother cells of this species, plastids duplicated their own DNA prior to the plastidkinesis of the first plastid division, but did not replicate plastid DNA prior to the plastidkinesis of the second plastid division. Therefore, the DNA content of those plastids in which division had been completed was reduced to half its initial value. This indicates that the DNA replication pattern of plastids in spore mother cells corresponds to that of cell nuclei during premeiosis and meiosis inA. punctatus.     

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.     

Heterogeneity of chloroplasts in Acetabularia mediterranea. Heterogeneous distribution and morphology of chloroplast DNA

A high percentage of chloroplasts in the siphonaceous green alga Acetabularia mediterranea lacks DNA: staining with the sensitive DNA-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) revealed that DNA was present only in 47–51% of the total plastid population. The distribution of DNA-containing chloroplasts appeared heterogeneous, exhibiting an apicobasal gradient. DNA could be detected in 64, 46, 35, and 36% of the plastids from the apical, subapical, middle, and basal part of the cell, respectively. DNA morphology also exhibited heterogeneity. Three types of nucleoid were distinguished: (1) round compact nucleoids; (2) long thin nucleoids characteristic of chloroplasts of the cell apex and the whorls; and (3) elaborate nucleoids appearing to consist of several subunits, which were more typical of the middle and basal part of the cell. On the basis of the nucleoid morphology and the decrease in DNA-containing plastids from the apex towards the basis of the cell, we propose a model for the development of plastids lacking DNA in relation to chloroplast replication.     

CHLOROPLAST DEVELOPMENT DURING SPOROGENESIS IN SIX SPECIES OF MOSSES

Chloroplast development during sporogenesis in Mnium cuspidatum, M. medium, M. rostratum, Aulacomnium heterostichum, Bartramia pomiformis, and Timmia megapolitana is as follows: During the early mitotic divisions in the sporogenous area of the capsule the number of plastids is reduced from many to one cup-shaped plastid per sporogenous cell. This single plastid divides during the early spore-mother-cell stage. A second division of plastids produces four plastids within each spore-mother-cell. A massive accumulation of starch occurs within each of the four plastids. Following meiosis, the single plastid allocated to each spore produces distinct lobes that are “blebbed” off as proplastids. A photosynthetic membrane system is established within the many proplastids as each spore matures.     

Was the evolution of plastid genetic machinery discontinuous?

The plastid nucleoid consists of plastid DNA and various, mostly uncharacterized, DNA-binding proteins. The plastid DNA undoubtedly originated from an ancestral cyanobacterial genome, but the origin of the nucleoid proteins appears complex. Initial biochemical analysis of these proteins, as well as comparative genome informatics, suggest that proteins of eukaryotic origin replaced most of the original prokaryotic proteins during the evolution of plastids in the lineage of green plants.     

CYTOCHEMICAL STUDIES ON PROCHLOROPHYTES: LOCALIZATION OF DNA AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE-OXYGENASE1

We studied the distribution of the DNA-containing region and the ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCo) content of polyhedral bodies in three different prochlorophyte cell types in a search for broad evolutionary affinities of these chlorophyll b-containing prokaryotes. DNA was localized by DAPI staining and electron microscopy utilizing monoclonal anti-DNA antibody 2C-10 plus a secondary antibody labeled with colloidal gold. Antibodies against the large RuBisCo subunit from a higher plant raised in rabbits were used to localize RuBisCo in polyhedral bodies. We studied Prochloron Lewin cells from two different didemnid ascidian hosts (Lissoclinum patella and Didemnum molle) collected in Palau, West Caroline Islands, and cells of Prochlorothrix hollandica Burger-Wiersma, Stal, and Mur grown in laboratory culture. Cells of the blue-green alga Anabaena 7120 were studied for comparison. The DNA distribution was markedly different in the two Prochloron cell types. The thylakoids in cells from L. patella were concentrically arranged around a large central vacuole; the DNA-containing stromal areas appeared in thin sections as a concentric arcs between the thylakoid stacks. The central vacuole was lacking in cells from D. molle, and the thylakoid stacks and strands of DNA-containing stroma showed a more haphazard arrangement. In the filamentous Prochlorothrix the DNA-containing stroma was largely limited to a central nucleoid structure running the length of the cell. Although the DNA arrangements in Prochloron might be considered “chloroplast-like” since DNA-containing stroma is distributed, as in chloroplasts, in scattered sites among photosynthetic membranes, this is not so in Prochlorothrix, where there is an axial nucleoid, as in many filamentous cyanobacteria. Our anti-RuBisCo antibodies were selectively bound to the polyhedral bodies of all three cell types, indicating that Prochloron and Prochlorothrix, like many other autotrophic prokaryotes, possess typical carboxysomes.     

Fates of Evolutionarily Distinct,Plastid‐type Glyceraldehyde 3‐phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome—one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte‐derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid‐type glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte‐derived tertiary plastids: “gapC1h” acquired from the haptophyte endosymbiont and “gapC1p” inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid‐type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT‐derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.