Plastid genome sequencing,comparative genomics,and phylogenomics: Current status and prospects

Abstract More than 190 plastid genomes have been completely sequenced during the past two decades due to advances in DNA sequencing technologies. Based on this unprecedented abundance of data, extensive genomic changes have been revealed in the plastid genomes. Inversion is the most common mechanism that leads to gene order changes. Several inversion events have been recognized as informative phylogenetic markers, such as a 30‐kb inversion found in all living vascular plants minus lycopsids and two short inversions putatively shared by all ferns. Gene loss is a common event throughout plastid genome evolution. Many genes were independently lost or transferred to the nuclear genome in multiple plant lineages. The trnR‐CCG gene was lost in some clades of lycophytes, ferns, and seed plants, and all the ndh genes were absent in parasitic plants, gnetophytes, Pinaceae, and the Taiwan moth orchid. Certain parasitic plants have, in particular, lost plastid genes related to photosynthesis because of the relaxation of functional constraint. The dramatic growth of plastid genome sequences has also promoted the use of whole plastid sequences and genomic features to solve phylogenetic problems. Chloroplast phylogenomics has provided additional evidence for deep‐level phylogenetic relationships as well as increased phylogenetic resolutions at low taxonomic levels. However, chloroplast phylogenomics is still in its infant stage and rigorous analysis methodology has yet to be developed.     

Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.     

Functional gene losses occur with minimal size reduction in the plastid genome of the parasitic liverwort Aneura mirabilis

Aneura mirabilis is a parasitic liverwort that exploits an existing mycorrhizal association between a basidiomycete and a host tree. This unusual liverwort is the only known parasitic seedless land plant with a completely nonphotosynthetic life history. The complete plastid genome of A. mirabilis was sequenced to examine the effect of its nonphotosynthetic life history on plastid genome content. Using a partial genomic fosmid library approach, the genome was sequenced and shown to be 108,007 bp with a structure typical of green plant plastids. Comparisons were made with the plastid genome of Marchantia polymorpha, the only other liverwort plastid sequence available. All ndh genes are either absent or pseudogenes. Five of 15 psb genes are pseudogenes, as are 2 of 6 psa genes and 2 of 6 pet genes. Pseudogenes of cysA, cysT, ccsA, and ycf3 were also detected. The remaining complement of genes present in M. polymorpha is present in the plastid of A. mirabilis with intact open reading frames. All pseudogenes and gene losses co-occur with losses detected in the plastid of the parasitic angiosperm Epifagus virginiana, though the latter has functional gene losses not found in A. mirabilis. The plastid genome sequence of A. mirabilis represents only the second liverwort, and first mycoheterotroph, to have its plastid genome sequenced. We observed a pattern of genome evolution congruent with functional gene losses in parasitic angiosperms but suggest that its plastid genome represents a genome in the early stages of decay following the relaxation of selection pressures.     

The tobacco plastid accD gene is essential and is required for leaf development

Angiosperm plastid genomes typically encode approximately 80 polypeptides, mainly specifying plastid-localized functions such as photosynthesis and gene expression. Plastid protein synthesis and expression of the plastid clpP1 gene are essential for development in tobacco, indicating the presence of one or more plastid genes whose influence extends beyond the plastid compartment. The plastid accD gene encodes the beta-carboxyl transferase subunit of acetyl-CoA carboxylase and is present in the plastids of most flowering plants, including non-photosynthetic parasitic plants. We replaced the wild-type accD gene with an aadA-disrupted mutant allele using homologous recombination. Persistent heteroplasmy in the presence of antibiotics indicated that the wild-type accD allele was essential. The phenotype of the accD knockout was revealed in plastid transformants grown in the absence of antibiotics. Leaves contained pale green sectors and lacked part or all of the leaf lamina due to arrested division or loss of cells. Abnormal structures were present in plastids found in mutant plants, indicating that accD might be required to maintain the plastid compartment. Loss of the plastid compartment would be expected to be lethal. These results provide genetic evidence showing the essential role of plastid ACCase in the pathway leading to the synthesis of products required for the extra-plastidic processes needed for leaf development.     

A subset of conserved tRNA genes in plastid DNA of nongreen plants.

The plastid genome of the nonphotosynthetic parasitic plant Epifagus virginiana contains only 17 of the 30 tRNA genes normally found in angiosperm plastid DNA. Although this is insufficient for translation, the genome is functional, so import of cytosolic tRNAs into plastids has been suggested. This raises the question of whether the tRNA genes that remain in E. virginiana plastid DNA are active or have just fortuitously escaped deletion. We report the sequences of 20 plastid tRNA loci from Orobanche minor, which shares a nonphotosynthetic ancestor with E. virginiana. The two species have 9 intact tRNA genes in common, the others being defunct in one or both species. The intron-containing trnLUAA gene is absent from E. virginiana, but it is intact, transcribed, and spliced in O. minor. The shared intact genes are better conserved than intergenic sequences, which indicates that these genes are being maintained by natural selection and, therefore, must be functional. For the most part, the tRNA species conserved in nonphotosynthetic plastids are also those that have never been found to be imported in plant mitochondria, which suggests that the same rules may govern tRNA import in the two organelles. A small photosynthesis gene, psbI, is still intact in O. minor, and computer simulations show that some small nonessential genes have an appreciable chance of escaping deletion.     

Plastid translation and transcription genes in a non-photosynthetic plant: intact, missing and pseudo genes

The non-photosynthetic, parasitic flowering plant Epifagus virginiana has recently been shown to contain a grossly reduced plastid genome that has lost many photosynthetic and chloro-respiratory genes. We have cloned and sequenced a 3.9 kb domain of plastid DNA from Epifagus to investigate the patterns of evolutionary change in such a reduced genome and to determine which genes are still present and likely to be functional. This 3.9 kb domain is colinear with a 35.4 kb region of tobacco chloroplast DNA, differing from it by a minimum of 11 large deletions varying in length from 354 bp to 11.5 kb, as well as by a number of small deletions and insertions. The nine genes retained in Epifagus encode seven tRNAs and two ribosomal proteins and are coextensive and highly conserved in sequence with homologs in photosynthetic plants. This suggests that these genes are functional in Epifagus and, together with evidence that the Epifagus plastid genome is transcribed, implies that plastid gene products play a role in processes other than photosynthesis and gene expression. Genes that are completely absent include not only photosynthetic genes, but surprisingly, genes encoding three subunits of RNA polymerase, four tRNAs and one ribosomal protein. In addition, only pseudogenes are found for two other tRNAs. Despite these defunct tRNA genes, codon and amino acid usage in Epifagus protein genes is normal. We therefore hypothesize that the expression of plastid genes in Epifagus relies on the import of nuclear encoded tRNAs and RNA polymerase from the cytoplasm.     

Evolution of the chloroplast genome

We discuss the suggestion that differences in the nucleotide composition between plastid and nuclear genomes may provide a selective advantage in the transposition of genes from plastid to nucleus. We show that in the adenine, thymine (AT)-rich genome of Borrelia burgdorferi several genes have an AT-content lower than the average for the genome as a whole. However, genes whose plant homologues have moved from plastid to nucleus are no less AT-rich than genes whose plant homologues have remained in the plastid, indicating that both classes of gene are able to support a high AT-content. We describe the anomalous organization of dinoflagellate plastid genes. These are located on small circles of 2-3 kbp, in contrast to the usual plastid genome organization of a single large circle of 100-200 kbp. Most circles contain a single gene. Some circles contain two genes and some contain none. Dinoflagellate plastids have retained far fewer genes than other plastids. We discuss a similarity between the dinoflagellate minicircles and the bacterial integron system.     

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.     

Nucleus-encoded genes for plastid-targeted proteins in Helicosporidium: functional diversity of a cryptic plastid in a parasitic alga

Plastids are the organelles of plants and algae that house photosynthesis and many other biochemical pathways. Plastids contain a small genome, but most of their proteins are encoded in the nucleus and posttranslationally targeted to the organelle. When plants and algae lose photosynthesis, they virtually always retain a highly reduced "cryptic" plastid. Cryptic plastids are known to exist in many organisms, although their metabolic functions are seldom understood. The best-studied example of a cryptic plastid is from the intracellular malaria parasite, Plasmodium, which has retained a plastid for the biosynthesis of fatty acids, isoprenoids, and heme by the use of plastid-targeted enzymes. To study a completely independent transformation of a photosynthetic plastid to a cryptic plastid in another alga-turned-parasite, we conducted an expressed sequence tag (EST) survey of Helicosporidium. This parasite has recently been recognized as a highly derived green alga. Based on phylogenetic relationships to other plastid homologues and the presence of N-terminal transit peptides, we have identified 20 putatively plastid-targeted enzymes that are involved in a wide variety of metabolic pathways. Overall, the metabolic diversity of the Helicosporidium cryptic plastid exceeds that of the Plasmodium plastid, as it includes representatives of most of the pathways known to operate in the Plasmodium plastid as well as many others. In particular, several amino acid biosynthetic pathways have been retained, including the leucine biosynthesis pathway, which was only recently recognized in plant plastids. These two parasites represent different evolutionary trajectories in plastid metabolic adaptation.     

Purification of Plastids from the Dinoflagellate Lingulodinium

Peridinin-containing dinoflagellates are a group of generally marine and photosynthetic protists whose plastids display a number of unusual features. In particular, the plastid genome may be reduced to as few as a dozen genes, and it is not clear if all these genes are expressed. To begin to characterize the plastid proteins, we attempted to purify chloroplasts from the dinoflagellate Lingulodinium polyedrum. We tested several different protocols and found that the organelles were inherently fragile and difficult to isolate intact. In particular, standard purification protocols as described for higher plants produced only broken plastids, as judged by complete loss of the stromal protein RuBisCO. We found that small amounts of RuBisCO could be retained in the plastids if the cells were treated with cellulase prior to lysis. Finally, we report that almost all RuBisCO was retained in plastids prepared from cells subjected to a heat shock treatment, although cellular proteins were denatured by the treatment.     

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales)

Premise

Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome.

Methods

We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω).

Results

Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK.

Conclusions

We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.     

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.     

Hypothesis: Gene‐rich plastid genomes in red algae may be an outcome of nuclear genome reduction

Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non‐photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage.     

THE EVOLUTION OF RNA POLYMERASE II IN RED ALGAE

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.     

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.     

Migration of the plastid genome to the nucleus in a peridinin dinoflagellate

Dinoflagellate algae are important primary producers and of significant ecological and economic impact because of their ability to form "red tides". They are also models for evolutionary research because of an unparalleled ability to capture photosynthetic organelles (plastids) through endosymbiosis. The nature and extent of the plastid genome in the dominant perdinin-containing dinoflagellates remain, however, two of the most intriguing issues in plastid evolution. The plastid genome in these taxa is reduced to single-gene minicircles encoding an incomplete (until now 15) set of plastid proteins. The location of the remaining photosynthetic genes is unknown. We generated a data set of 6,480 unique expressed sequence tags (ESTs) from the toxic dinoflagellate Alexandrium tamarense (for details, see the Experimental Procedures in the Supplemental Data) to find the missing plastid genes and to understand the impact of endosymbiosis on genome evolution. Here we identify 48 of the non-minicircle-encoded photosynthetic genes in the nuclear genome of A. tamarense, accounting for the majority of the photosystem. Fifteen genes that are always found on the plastid genome of other algae and plants have been transferred to the nucleus in A. tamarense. The plastid-targeted genes have red and green algal origins. These results highlight the unique position of dinoflagellates as the champions of plastid gene transfer to the nucleus among photosynthetic eukaryotes.     

Why are plastid genomes retained in non-photosynthetic organisms?

The evolution of the plastid from a photosynthetic bacterial endosymbiont involved a dramatic reduction in the complexity of the plastid genome, with many genes either discarded or transferred to the nucleus of the eukaryotic host. However, this evolutionary process has not gone to completion and a subset of genes remains in all plastids examined to date. The various hypotheses put forward to explain the retention of the plastid genome have tended to focus on the need for photosynthetic organisms to retain a genetic system in the chloroplast, and they fail to explain why heterotrophic plants and algae, and the apicomplexan parasites all retain a genome in their non-photosynthetic plastids. Here we consider two additional explanations: the 'essential tRNAs' hypothesis and the 'transfer-window' hypothesis.     

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 when photosynthesis comes under full control of the "host" nucleus (e.g., as in heterokonts, haptophytes, and euglenophytes). Genes presently found in the nucleomorph seem to be restricted to those involved in its own maintenance and to that of the plastid; other genes were lost as the endosymbiont was progressively reduced to its present state. Surprisingly, we found that the cryptophyte Pyrenomonas helgolandii possesses a novel type of actin gene that 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 which are unrelated to plastid function. These genes are akin to the products of gene duplication or lateral transfer and provide a source of evolutionary novelty that can 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.     

Mechanisms of Functional and Physical Genome Reduction in Photosynthetic and Nonphotosynthetic Parasitic Plants of the Broomrape Family

Nonphotosynthetic plants possess strongly reconfigured plastomes attributable to convergent losses of photosynthesis and housekeeping genes, making them excellent systems for studying genome evolution under relaxed selective pressures. We report the complete plastomes of 10 photosynthetic and nonphotosynthetic parasites plus their nonparasitic sister from the broomrape family (Orobanchaceae). By reconstructing the history of gene losses and genome reconfigurations, we find that the establishment of obligate parasitism triggers the relaxation of selective constraints. Partly because of independent losses of one inverted repeat region, Orobanchaceae plastomes vary 3.5-fold in size, with 45 kb in American squawroot (Conopholis americana) representing the smallest plastome reported from land plants. Of the 42 to 74 retained unique genes, only 16 protein genes, 15 tRNAs, and four rRNAs are commonly found. Several holoparasites retain ATP synthase genes with intact open reading frames, suggesting a prolonged function in these plants. The loss of photosynthesis alters the chromosomal architecture in that recombinogenic factors accumulate, fostering large-scale chromosomal rearrangements as functional reduction proceeds. The retention of DNA fragments is strongly influenced by both their proximity to genes under selection and the co-occurrence with those in operons, indicating complex constraints beyond gene function that determine the evolutionary survival time of plastid regions in nonphotosynthetic plants.