Klaus V. Kowallik

Gene transfer to the nucleus and the evolution of chloroplasts

Photosynthetic eukaryotes, particularly unicellular forms, possess a fossil record that is either wrought with gaps or difficult to interpret, or both. Attempts to reconstruct their evolution have focused on plastid phylogeny, but were limited by the amount and type of phylogenetic information contained within single genes. Among the 210 different protein-coding genes contained in the completely sequenced chloroplast genomes from a glaucocystophyte, a rhodophyte, a diatom, a euglenophyte and five land plants, we have now identified the set of 45 common to each and to a cyanobacterial outgroup genome. Phylogenetic inference with an alignment of 11,039 amino-acid positions per genome indicates that this information is sufficient — but just barely so — to identify the rooted nine-taxon topology. We mapped the process of gene loss from chloroplast genomes across the inferred tree and found that, surprisingly, independent parallel gene losses in multiple lineages outnumber phylogenetically unique losses by more than 4:1. We identified homologues of 44 different plastid-encoded proteins as functional nuclear genes of chloroplast origin, providing evidence for endosymbiotic gene transfer to the nucleus in plants.

The chloroplast genome of a chlorophylla+c-containing alga,Odontella sinensis

The chloroplast genome of a marine centric diatom,Odontella sinensis, was cloned and sequenced. The circular genome is 119,704 bp in length (AC=Z67753;). It contains an inverted repeat sequence of 7,725 bp separating two single-copy regions of 38,908 and 65,346 bp, respectively, and 174 genes and open reading frames, of which nine are duplicated within the inverted repeat segments.

A nuclear‐encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana

To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear‐ and plastome‐encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome‐encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T‐DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark‐grown seedlings and its levels do not increase dramatically during light‐induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo‐ and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial‐like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.

Genes of Cyanobacterial Origin in Plant Nuclear Genomes Point to a Heterocyst-Forming Plastid Ancestor

Plastids are descended from a cyanobacterial symbiosis which occurred over 1.2 billion years ago. During the course of endosymbiosis, most genes were lost from the cyanobacteriums genome and many were relocated to the host nucleus through endosymbiotic gene transfer (EGT). The issue of how many genes were acquired through EGT in different plant lineages is unresolved. Here, we report the genome-wide frequency of gene acquisitions from cyanobacteria in 4 photosynthetic eukaryotes--Arabidopsis, rice, Chlamydomonas, and the red alga Cyanidioschyzon--by comparison of the 83,138 proteins encoded in their genomes with 851,607 proteins encoded in 9 sequenced cyanobacterial genomes, 215 other reference prokaryotic genomes, and 13 reference eukaryotic genomes. The analyses entail 11,569 phylogenies inferred with both maximum likelihood and Neighbor-Joining approaches. Because each phylogenetic result is dependent not only upon the reconstruction method but also upon the site patterns in the underlying alignment, we investigated how the reliability of site pattern generation via alignment affects our results: if the site patterns in an alignment differ depending upon the order in which amino acids are introduced into multiple sequence alignment--N- to C-terminal versus C- to N-terminal--then the phylogenetic result is likely to be artifactual. Excluding unreliable alignments by this means, we obtain a conservative estimate, wherein about 14% of the proteins examined in each plant genome indicate a cyanobacterial origin for the corresponding nuclear gene, with higher proportions (17-25%) observed among the more reliable alignments. The identification of cyanobacterial genes in plant genomes affords access to an important question: from which type of cyanobacterium did the ancestor of plastids arise? Among the 9 cyanobacterial genomes sampled, Nostoc sp. PCC7120 and Anabaena variabilis ATCC29143 were found to harbor collections of genes which are-in terms of presence/absence and sequence similarity-more like those possessed by the plastid ancestor than those of the other 7 cyanobacterial genomes sampled here. This suggests that the ancestor of plastids might have been an organism more similar to filamentous, heterocyst-forming (nitrogen-fixing) representatives of section IV recognized in Staniers cyanobacterial classification. Members of section IV are very common partners in contemporary symbiotic associations involving endosymbiotic cyanobacteria, which generally provide nitrogen to their host, consistent with suggestions that fixed nitrogen supplied by the endosymbiont might have played an important role during the origin of plastids.

Annotated English translation of Mereschkowsky's 1905 paper ‘Über Natur und Ursprung der Chromatophoren im Pflanzenreiche’

That plastids were once free-living cyanobacteria is now taken for granted by many, and for good reasons, for there is a wealth of data – in particular from the comparison of plastid and cyanobacterial genomes – that support this view. There is currently no seriously entertained alternative hypothesis to the view that plastids descend from cyanobacteria. But that was not always the case. Well into the 1970s there was a generally favoured alternative hypothesis, namely that early in evolution plastids arose de novo from within a non-plastid bearing cell (an autogenous origin) rather than through invasion by a cyanobacterium into a non-plastid-bearing cell with subsequent intracellular coexistence and reduction to an organelle (an endosymbiotic origin). Interestingly, the shift from autogenous to endosymbiotic hypotheses during the 1970s was a reversal of state for during the first two decades of this century, the endosymbiont hypothesis for the origins of plastids (and mitochondria, which will not be further discussed here) was very popular among biologists. It fell into disfavour shortly after the First World War, for reasons that are very difficult to summarize briefly, and remained scorned for 50 years (see Sapp, 1994, for an historical account in English, and Hoxtermann, 1998, for a succinct historical account in German). So where did the first version of the endosymbiont hypothesis come from? In a nutshell, it came from Konstantin Sergejewiz Merezkovskij (usually written as Constantin Mereschkowsky), a Russian botanist of little standing who worked at a rather small and by no means prominent university in Kasan and who published a very remarkable paper in 1905. We are not aware of any true precedent for his paper, which draws upon three lines of evidence known at the time.

Gene-cluster analysis in chloroplast genomics

The work was supported by DFG grants Ko 439/6-1 through 439/6-3. We gratefully acknowledge our collaborators at Dusseldorf and W. Martin for helpful discussions.

Size, conformation and purity of chloroplast DNA of some higher plants

1. Chloroplast DNA of Antirrhinum majus, Oenothera hookeri, Beta vulgaris and Spinacia oleracea band at the same buoyant density of 1.697 g-cm-3 in neutral CsCl equilibrium gradients. The corresponding nuclear DNAs band at 1.691, 1.703, 1.695 and 1.695 g-cm-3, respectively. The purity of chloroplast and nuclear DNA can be assessed objectively only in the cases of Antirrhinum and Oenothera. 2. Electron microscopic analysis of chloroplast DNA, purified in CsCl or CsCl/ethidium bromide gradients, revealed up to 80% circular molecules. Of these about 15% were of supertwisted conformation. Best yields of circular molecules were recovered when populations of unbroken chloroplasts were subjected to DNAase and phosphodiesterase treatment, and when the DNA was purified from viscous lysates by centrifugation into a CsCl cushion. Treatment of plastids with DNAase alone did not guarantee complete degradation of nuclear DNA. 3. The average contour length of the open circular chloroplast DNA molecules was basically similar for all four plants. They were 45.9 plus or minus 2.1 mum for Antirrhinum, 45.7 plus or minus 1.9 mum for Spinacia, 44.9 plus or minus 1.7 mum for Beta and 45.2 mum for Oenothera. This is comparable to the size derived for the coding capacity of chloroplast DNA from reassociation experiments. As much as 15% of the total population of circles in chloroplast DNA of Spinacia were circular dimers.

A physical map of Nicotiana tabacum plastid DNA including the location of structural genes for ribosomal RNAs and the large subunit of ribulose bisphosphate carboxylase/oxygenase

Summary1)Tobacco plastids contain a homogeneous population of double-stranded circular DNA molecules, 101 Megadalton (160 kbp) in circumference. In neutral CsCl equilibrium gradients, this DNA displays a density of 1.697 g · cm−3 which is equivalent to an average base composition of 37.7 mole-% G+C.2)A restriction endonuclease fragment map of the tobacco plastid chromosome is presented for the enzymes Bgl I, Sal I, Xho I and Pvu II which together dissect the DNA molecule into about 60 fragments. The map was derived by sequential digestion employing the previously described Seaplaque technique. The tobacco plastid chromosome has an anatomy similar to that of many other higher plants; the circular DNA is segmentally organized into two unique sequence segments of approximately 24 and 95 kbp separated on each side by a large inverted duplication of at least 20.4 kbp.3)Saturation and blot hybridization showed that the genes for the 16S and 23S pt-rRNAs are duplicated. Each copy of the inverted repeat contains one set of rRNA genes; about 26 kbp (short distance) separate the sets from each other.4)Cloned fragments of spinach ptDNA nick translated to high specific activity in vitro were used to probe the location of the large subunit gene of ribulose bisphosphate carboxylase/oxygenase on tobacco ptDNA. A 3.5 kbp-long fragment of the large single-copy region of the tobacco chromosome is complementary to structural sequences of the spinach gene.5)Mapping and hybridization data suggest that the tobacco and spinach ptDNAs share striking similarities in anatomy and sequence.

Distribution and nomenclature of protein coding genes in 12 sequenced chloroplast genomes

Abbreviations: bind., binding; Chl, Chlorella vulgaris ;C pa,Cyanophora paradoxa ;E pi, Epifagus virginiana; Eug, Euglena gracilis; hom., homologue; Mar, Marchantia polymorpha ; Nic, Nicotiana tabacum; Odo, Odontella sinensis ;O ry,Oryza sativa ;P in,Pinus thunbergii; Pla, Plasmodium falciparum ;P or,Porphyra purpurea; prot., protein; RT, reverse transcriptase; sim., similar to; SU, subunit; Syn, Synechocystis sp. PCC6803; Zea, Zea mays

Substitutional editing of transcripts from genes of cyanobacterial origin in the dinoflagellate Ceratium horridum

Peridinin‐containing dinoflagellates, a group of alveolate organisms, harbour small plasmids called minicircles. As most of these minicircles encode genes of cyanobacterial origin, which are also found in plastid genomes of stramenopiles, they were thought to represent the plastid genome of peridinin‐containing dinoflagellates. The analyses of minicircle derived mRNAs and the 16S rRNA showed that extensive editing of minicircle gene transcripts is common for Ceratium horridum. Posttranscriptional changes occur predominantly by editing A into G, but other types of editing including a previously unreported A to C transversion were also detected. This leads to amino acid changes in most cases or, in one case, to the elimination of a stop‐codon. Interestingly, the edited mRNAs show higher identities to homologous sequences of other peridinin‐containing dinoflagellates than their genomic copy. Thus, our results imply that transcript editing of genes of cyanobacterial origin is species specific in peridinin‐containing dinoflagellates and demonstrate that editing of genes of cyanobacterial origin is not restricted to land plants.