Phylogeny of Dinoflagellate Plastid Genes Recently Transferred to the Nucleus Supports a Common Ancestry with Red Algal Plastid Genes

It is generally accepted that peridinin-containing dinoflagellate plastids are derived from red alga, but whether they are secondary plastids equivalent to plastids of stramenopiles, haptophytes, or cryptophytes, or are tertiary plastids derived from one of the other secondary plastids, has not yet been completely resolved. As secondary plastids, plastid gene phylogeny should mirror that of nuclear genes, while incongruence in the two phylogenies should be anticipated if their origin was as tertiary plastids. We have analyzed the phylogeny of plastid-encoded genes from Lingulodinium as well as that of nuclear-encoded dinoflagellate homologues of plastid-encoded genes conserved in all other plastid genome sequences. Our analyses place the dinoflagellate, stramenopile, haptophyte, and cryptophyte plastids firmly in the red algal lineage, and in particular, the close relationship between stramenopile plastid genes and their dinoflagellate nuclear-encoded homologues is consistent with the hypothesis that red algal-type plastids have arisen only once in evolution.     

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.     

A tertiary plastid uses genes from two endosymbionts

The origin and subsequent spread of plastids by endosymbiosis had a major environmental impact and altered the course of a great proportion of eukaryotic biodiversity. The ancestor of dinoflagellates contained a secondary plastid that was acquired in an ancient endosymbiotic event, where a eukaryotic cell engulfed a red alga. This is known as secondary endosymbiosis and has happened several times in eukaryotic evolution. Certain dinoflagellates, however, are unique in having replaced this secondary plastid in an additional (tertiary) round of endosymbiosis. Most plastid proteins are encoded in the nucleus of the host and are targeted to the organelle. When secondary or tertiary endosymbiosis takes place, it is thought that these genes move from nucleus to nucleus, so the plastid retains the same proteome. We have conducted large-scale expressed sequence tag (EST) surveys from Karlodinium micrum, a dinoflagellate with a tertiary haptophyte-derived plastid, and two haptophytes, Isochrysis galbana and Pavlova lutheri. We have identified all plastid-targeted proteins, analysed the phylogenetic origin of each protein, and compared their plastid-targeting transit peptides. Many plastid-targeted genes in the Karlodinium nucleus are indeed of haptophyte origin, but some genes were also retained from the original plastid (showing the two plastids likely co-existed in the same cell), in other cases multiple isoforms of different origins exist. We analysed plastid-targeting sequences and found the transit peptides in K.micrum are different from those found in either dinoflagellates or haptophytes, pointing to a plastid with an evolutionarily chimeric proteome, and a massive remodelling of protein trafficking during plastid replacement.     

Presequence Acquisition During Secondary Endocytobiosis and thePossible Role of Introns

Targeting of nucleus-encoded proteins into chloroplasts is mediated by N-terminal presequences. During evolution of plastids from formerly free-living cyanobacteria by endocytobiosis, genes for most plastid proteins have been transferred from the plastid genome to the nucleus and subsequently had to be equipped with such plastid targeting sequences. So far it is unclear how the gene domains coding for presequences and the respective mature proteins may have been assembled. While land plant plastids are supposed to originate from a primary endocytobiosis event (a prokaryotic cyanobacterium was taken up by a eukaryotic cell), organisms with secondary plastids like diatoms experienced a second endocytobiosis step involving a eukaryotic alga taken up by a eukaryotic host cell. In this group of algae, apparently most genes encoding chloroplast proteins have been transferred a second time (from the nucleus of the endosymbiont to the nucleus of the secondary host) and thus must have been equipped with additional targeting signals. We have analyzed cDNAs and the respective genomic DNA fragments of seven plastid preproteins from the diatom Phaeodactylum tricornutum. In all of these genes we found single spliceosomal introns, generally located within the region coding for the N-terminal plastid targeting sequences or shortly downstream of it. The positions of the introns can be related to the putative phylogenetic histories of the respective genes, indicating that the bipartite targeting sequences in these secondary algae might have evolved by recombination events via introns.The nucleotide sequences have been deposited at Genbank under accession numbers AY191862, AY191863, AY191864, AY191865, AY191866, AY191867, and AY191868.     

Rampant gene loss in the underground orchid Rhizanthella gardneri highlights evolutionary constraints on plastid genomes

Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on photosynthesis for energy and have retained ～110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer.     

Development of Molecular Probes for Dinophysis (Dinophyceae) Plastid: A Tool to Predict Blooming and Explore Plastid Origin

Dinophysis are species of dinoflagellates that cause diarrhetic shellfish poisoning. We have previously reported that they probably acquire plastids from cryptophytes in the environment, after which they bloom. Thus monitoring the intracellular plastid density in Dinophysis and the source cryptophytes occurring in the field should allow prediction of Dinophysis blooming. In this study the nucleotide sequences of the plastid-encoded small subunit ribosomal RNA gene and rbcL (encoding the large subunit of RuBisCO) from Dinophysis spp. were compared with those of cryptophytes, and genetic probes specific for the Dinophysis plastid were designed. Fluorescent in situ hybridization (FISH) showed that the probes bound specifically to Dinophysis plastids. Also, FISH on collected nanoplankton showed the presence of probe-hybridized eukaryotes, possibly cryptophytes with plastids identical to those of Dinophysis. These probes are useful not only as markers for plastid density and activity of Dinophysis, but also as tools for monitoring cryptophytes that may be sources of Dinophysis plastids.     

Exclusion of plastid nucleoids and ribosomes from stromules in tobacco and Arabidopsis

Stromules are stroma-filled tubules that extend from the surface of plastids and allow the transfer of proteins as large as 550 kDa between interconnected plastids. The aim of the present study was to determine if plastid DNA or plastid ribosomes are able to enter stromules, potentially permitting the transfer of genetic information between plastids. Plastid DNA and ribosomes were marked with green fluorescent protein (GFP) fusions to LacI, the lac repressor, which binds to lacO-related sequences in plastid DNA, and to plastid ribosomal proteins Rpl1 and Rps2, respectively. Fluorescence from GFP-LacI co-localised with plastid DNA in nucleoids in all tissues of transgenic tobacco (Nicotiana tabacum L.) examined and there was no indication of its presence in stromules, not even in hypocotyl epidermal cells, which contain abundant stromules. Fluorescence from Rpl1-GFP and Rps2-GFP was also observed in a punctate pattern in chloroplasts of tobacco and Arabidopsis [Arabidopsis thaliana (L.) Heynh.], and fluorescent stromules were not detected. Rpl1-GFP was shown to assemble into ribosomes and was co-localised with plastid DNA. In contrast, in hypocotyl epidermal cells of dark-grown Arabidopsis seedlings, fluorescence from Rpl1-GFP was more evenly distributed in plastids and was observed in stromules on a total of only four plastids (<0.02% of the plastids observed). These observations indicate that plastid DNA and plastid ribosomes do not routinely move into stromules in tobacco and Arabidopsis, and suggest that transfer of genetic information by this route is likely to be a very rare event, if it occurs at all.     

Genome evolution of a tertiary dinoflagellate plastid

The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata.     

Nucleus‐encoded mRNAs for Chloroplast Proteins GapA,PetA, and PsbO are Trans‐spliced in the Flagellate Euglena gracilis Irrespective of Light and Plastid Function

Euglena gracilis is a fresh‐water flagellate possessing secondary chloroplasts of green algal origin. In contrast with organisms possessing primary plastids, mRNA levels of nucleus‐encoded genes for chloroplast proteins in E. gracilis depend on neither light nor plastid function. However, it remains unknown, if all these mRNAs are trans‐spliced and possess spliced leader sequence at the 5′‐end and if trans‐splicing depends on light or functional plastids. This study revealed that polyadenylated mRNAs encoding the chloroplast proteins glyceraldehyde‐3‐phosphate dehydrogenase (GapA), cytochrome f (PetA), and subunit O of photosystem II (PsbO) are trans‐spliced irrespective of light or plastid function.     

EVOLUTIONARY ANALYSES OF THE NUCLEAR‐ENCODED PHOTOSYNTHETIC GENE psbO FROM TERTIARY PLASTID‐CONTAINING ALGAE IN DINOPHYTA1

Although the dinophytes generally possess red‐algal‐derived secondary plastids, tertiary plastids originating from haptophyte and diatom ancestors are recognized in some lineages within the Dinophyta. However, little is known about the nuclear‐encoded genes of plastid‐targeted proteins from the dinophytes with diatom‐derived tertiary plastids. We analyzed the sequences of the nuclear psbO gene encoding oxygen‐evolving enhancer protein from various algae with red‐algal‐derived secondary and tertiary plastids. Based on our sequencing of 10 new genes and phylogenetic analysis of PsbO amino acid sequences from a wide taxon sampling of red algae and organisms with red‐algal‐derived plastids, dinophytes form three separate lineages: one composed of peridinin‐containing species with secondary plastids, and the other two having haptophyte‐ or diatom‐derived tertiary plastids and forming a robust monophyletic group with haptophytes and diatoms, respectively. Comparison of the N‐terminal sequences of PsbO proteins suggests that psbO genes from a dinophyte with diatom‐derived tertiary plastids (Kryptoperidinium) encode proteins that are targeted to the diatom plastid from the endosymbiotic diatom nucleus as in the secondary phototrophs, whereas the fucoxanthin‐containing dinophytes (Karenia and Karlodinium) have evolved an additional system of psbO genes for targeting the PsbO proteins to their haptophyte‐derived tertiary plastids from the host dinophyte nuclei.     

Origin of the plastid in the anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta) as inferred from phylogenetic analysis based on the gene encoding the large subunit of form I-type RuBisCO

The dinoflagellate Gymnodinium mikimotoi Miyake et Kominami ex Oda possesses an anomalously pigmented plastid which contains 19′‐hexanoyloxyfucoxanthin, 19′‐butanoyloxyfucoxanthin and fucoxanthin instead of peridinin as the major carotenoids. Previously, we have shown that the plastid of G. mikimotoi belongs to the rhodoplast lineage as inferred from phylogenetic analyses based on the amino acid sequences deduced from psbA and psaA and the nucleotide sequence of the plastid small subunit ribosomal RNA. Furthermore, in the present study, we cloned and sequenced an additional representative plastid gene, rbcL, encoding the large subunit of ribulose 1–5 bisphosphate carboxylase/oxygenase (RuBisCO LSU) from G. mikimotoi. The amino acid sequence deduced from the rbcL gene of G. mikimotoi apparently revealed the conventional form I RuBisCO LSU, which is present in most photosynthetic organisms, and not the divergent form II existing in typically pigmented dinofl age Nates with plastids containing peridinin as the main carotenoid. This finding supports the hypothesis that the origins of the plastids in G. mikimotoi and peridinin‐type dinoflagellates are not related to each other. Molecular phylogenetic analysis based on the amino acid sequence deduced from the rbcL gene further showed that the plastid of G. mikimotoi belongs to the rhodoplast lineage. In particular, G. mikimotoi clustered with haptophytes in the phylogenetic tree. From this result, two hypotheses with respect to the origin of the plastid in G. mikimotoi can be proposed: G. mikimotoi may have engulfed a haptophyte‐like cell (tertiary symbiosis) or englulfed a rhodophyte‐like cell that was closely related to the origin of the plastid in the haptophyte (secondary symbiosis).     

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms,Bacillariophyceae)

Mitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.     

Tentoxin effects onSorghum: The role of polyphenol oxidase

Summary In an ultrastructural and cytochemical study of tentoxin-treatedSorghum bicolor (L.) Moench, both bundle sheath and mesophyll plastids were severely affected, Plastids from chlorotic leaf areas lacked most internal membranes yet had plastid ribosomes and large fibrillar areas of plastid DNA. In recovered areas (mottled yellow and green), cells were found that had plastids of near-normal ultrastructure as well as the severely affected plastid-types found in chlorotic leaf areas. Polyphenol oxidase (PPO) cytochemistry of these mottled leaf areas indicated that all recovered mesophyll plastids had PPO whereas all the abnormal mesophyll plastids showed no activity. Because bundle sheath plastids ofSorghum have no PPO activity at any developmental stage, yet are affected by tentoxin, PPO cannot be uniquely affected by this toxin. We suggest that tentoxin may affect the transport of cytosolic proteins into the plastid.     

Plastid inheritance in the planktonic raphid pennate diatom Pseudo-nitzschia delicatissima (Bacillariophyceae)

Plastid inheritance was followed during sexual reproduction in the raphid pennate diatom Pseudo-nitzschia delicatissima, using rbcL haplotypes as plastid identification tools. Pseudo-nitzschia species are dioecious and show functional anisogamy with 'male' mating type+(PNd(+)) cells and 'female' PNd(-) cells. Vegetative cells possess two plastids. In P. delicatissima, meiosis results in two gametes that both contribute two plastids to the zygote. The latter initially contains four plastids, but during auxospore development two of these four seem to disappear, and the initial cell emerging from the auxospore appears to contain only two. Here we assessed if the plastids are inherited strictly unipaternally, strictly biparentally, or randomly. We traced the source of the plastids in the F(1) generation by using PNd(+) and PNd(-) parental strains with different rbcL genotypes, here denoted AA (homoplastidial, with two plastids of rbcL haplotype A) and BB (homoplastidial; two plastids of haplotype B). Results showed that 16 out of 96 strains raised each from single F(1) cells had retained two paternal (PNd(+)) plastids, 20 had two maternal (PNd(-)) plastids and the remaining 60 had one maternal and one paternal plastid. This pattern is in accordance with the hypothesis that either two of the four plastids are eliminated during auxospore formation, or that all plastids are retained in the auxospore and segregate in pairs joining at random during the first mitotic division of the initial cell. Heteroplastidic F(1)-strains retained the AB genotype throughout the vegetative phase of their life cycle. The finding that 60 out of 96 F(1) strains were heteroplastidial contrasts with an absence of such genotypes in our strains raised from single cells sampled in the Gulf of Naples.     

Genetic transformation of plastids of different Solanaceae species using tobacco cells as organelle hosts

The plastid genome of angiosperms represents an attractive target for genetic manipulations. However plastid transformation of higher plants, especially of agriculturally valuable crops is an extremely difficult problem. Transformation protocols developed for tobacco 15 years ago failed to produce similar results with more than a handful of other species so far. We have analyzed plastid transformability of remote cytoplasmic hybrids (cybrids) that combine nuclei of tobacco, an easily transformable species, and plastids of some other, recalcitrant Solanaceae species. Here, we demonstrate that the plastids of five species of Solanaceae family, representing two subfamilies and three tribes, can be easily transformed if the plastids of these species are transferred into a cell of a transformable species (tobacco). The results can be considered to be an alternative approach to the development of plastid transformation technologies for recalcitrant species using a transformable intermediary (“clipboard”) host.     

Populations of plastids and mitochondria during male reproductive cell maturation in Nicotiana tabacum L.: A cytological basis for occasional biparental inheritance

The dynamics of plastid and mitochondrial populations in male reproductive cells of tobacco (Nicotiana tabacum L.) were examined during development using serial ultrathin sections and transmission electron microscopy to reconstruct 58 generative cells and 31 sperm cells at selected stages of maturation from generative cell formation through gametic fusion. The first haploid mitosis resulted in incomplete exclusion of plastids providing an average of 2.81 plastids and 82.7 mitochondria for each newly formed generative cell. During generative-cell maturation, plastid content decreased to an average of 0.48 plastids/generative cell at anthesis owing to autophagy of organelles. Plastids were present in low frequency within generative and sperm cells in the pollen tube and appeared to be transmitted, according to observations immediately prior to fertilization. This forms a cytological basis for genetic reports of occasional biparental plastid inheritance. In contrast, mitochondria were transmitted in larger numbers, and approximately 80 mitochondria per generative cell or sperm cell pair were retained throughout development. This provides a potentially stable source for the transmission of male mitochondrial DNA, if present at fertilization.Abbreviations GC generative cell - SC sperm cell We thank Dr. Frank J. Sonleitner, for helpful suggestions on the statistical calculations and Dr. Bing-Quan Huang for technical assistance in the preparation of embryo sacs during fertilization. This research was supported in part by U.S. Department of Agriculture grant 91-37304-6471. We gratefully acknowledge use of the Samuel Roberts Noble Electron Microscopy Laboratory of the University of Oklahoma.     

The Origin of Chlorarachniophyte Plastids, as Inferred from Phylogenetic Comparisons of Amino Acid Sequences of EF-Tu

A molecular phylogenetic analysis of elongation factor Tu (EF-Tu) proteins from plastids was performed in an attempt to identify the origin of chlorarachniophyte plastids, which are considered to have evolved from the endosymbiont of a photosynthetic eukaryote. Partial sequences of the genes for plastid EF-Tu proteins (1,080–1,089 bp) were determined for three algae that contain chlorophyll b, namely, Gymnochlora stellata (Chlorarachniophyceae), Bryopsis maxima (Ulvophyceae), and Pyramimonas disomata (Prasinophyceae). The deduced amino acid sequences were used to construct phylogenetic trees of the plastid and bacterial EF-Tu proteins by the maximum likelihood, the maximum parsimony, and the neighbor joining methods. The trees obtained in the present analysis suggest that all plastids that contain chlorophyll b are monophyletic and that the chlorarachniophyte plastids are closely related to those of the Ulvophyceae. The phylogenetic trees also suggest that euglenophyte plastids are closely related to prasinophycean plastids. The results indicate that the chlorarachniophyte plastids evolved from a green algal endosymbiont that was closely related to the Ulvophyceae and that at least two secondary endosymbiotic events have occurred in the lineage of algae with plastids that contain chlorophyll b. Received: 10 March 1997 / Accepted: 28 July 1997     

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots ( Arabidopsis , broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.