Wolfgang Löffelhardt

Cyanophora paradoxa Genome Elucidates Origin of Photosynthesis in Algae and Plants

Plastid Origins The glaucophytes, represented by the alga Cyanophora paradoxa, are the putative sister group of red and green algae and plants, which together comprise the founding group of photosynthetic eukaryotes, the Plantae. In their analysis of the genome of C. paradoxa, Price et al. (p. 843; see the Perspective by Spiegel) demonstrate a unique origin for the plastid in the ancestor of this supergroup, which retains much of the ancestral diversity in genes involved in carbohydrate metabolism and fermentation, as well as in the gene content of the mitochondrial genome. Moreover, about 3.3% of nuclear genes in C. paradoxa seem to carry a signal of cyanobacterial ancestry, and key genes involved in starch biosynthesis are derived from energy parasites such as Chlamydiae. Rapid radiation, reticulate evolution via horizontal gene transfer, high rates of gene divergence, loss, and replacement, may have diffused the evolutionary signals within this supergroup, which perhaps explains previous difficulties in resolving its evolutionary history. An ancient algal genome suggests a unique origin of the plastid in the ancestor to plants, algae, and glaucophytes. The primary endosymbiotic origin of the plastid in eukaryotes more than 1 billion years ago led to the evolution of algae and plants. We analyzed draft genome and transcriptome data from the basally diverging alga Cyanophora paradoxa and provide evidence for a single origin of the primary plastid in the eukaryote supergroup Plantae. C. paradoxa retains ancestral features of starch biosynthesis, fermentation, and plastid protein translocation common to plants and algae but lacks typical eukaryotic light-harvesting complex proteins. Traces of an ancient link to parasites such as Chlamydiae were found in the genomes of C. paradoxa and other Plantae. Apparently, Chlamydia-like bacteria donated genes that allow export of photosynthate from the plastid and its polymerization into storage polysaccharide in the cytosol.

Nucleotide sequence of the cyanelle genome fromCyanophora paradoxa

The complete nucleotide sequence of the cyanelle genome ofCyanophora paradoxa Pringsheim strain LB 555 was determined (accession number U30821). The circular molecule is 135,599 base pairs in length. The physical map of this DNA molecule is shown along with identified genes and open reading frames.

Protein import into cyanelles and complex chloroplasts

Higher-plant, green and red algal chloroplasts are surrounded by a double membrane envelope. The glaucocystophyte plastid (cyanelle) has retained a prokaryotic cell wall between the two envelope membranes. The complex chloroplasts of Euglena and dinoflagellates are surrounded by three membranes while the complex chloroplasts of chlorarachniophytes, cryptomonads, brown algae, diatoms and other chromophytes, are surrounded by 4 membranes. The peptidoglycan layer of the cyanelle envelope and the additional membranes of complex chloroplasts provide barriers to chloroplast protein import not present in the simpler double membrane chloroplast envelope. Analysis of presequence structure and in vitro import experiments indicate that proteins are imported directly from the cytoplasm across the two envelope membranes and peptidoglycan layer into cyanelles. Protein import into complex chloroplasts is however fundamentally different. Analysis of presequence structure and in vitro import into microsomal membranes has shown that translocation into the ER is the first step for protein import into complex chloroplasts enclosed by three or four membranes. In vivo pulse chase experiments and immunoelectronmicroscopy have shown that in Euglena, proteins are transported from the ER to the Golgi apparatus prior to import across the three chloroplast membranes. Ultrastructural studies and the presence of ribosomes on the outermost of the four envelope membranes suggests protein import into 4 membrane-bounded complex chloroplasts is directly from the ER like outermost membrane into the chloroplast. The fundamental difference in import mechanisms, post-translational direct chloroplast import or co-translational translocation into the ER prior to chloroplast import, appears to reflect the evolutionary origin of the different chloroplast types. Chloroplasts with a two-membrane envelope are thought to have evolved through the primary endosymbiotic association between a eukaryotic host and a photosynthetic prokaryote while complex chloroplasts are believed to have evolved through a secondary endosymbiotic association between a heterotrophic or possibly phototrophic eukaryotic host and a photosynthetic eukaryote.

The Cyanelles of Cyanophora Paradoxa

Abstract 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 seque...

IN VITRO IMPORT OF PRE-FERREDOXIN-NADP+-OXIDOREDUCTASE FROM CYANOPHORA PARADOXA INTO CYANELLES AND INTO PEA CHLOROPLASTS

Using a novel cyanelle isolation procedure we showed that pre‐ferredoxin‐NADP+‐oxidoreductase (pre‐FNR) from C. paradoxa is translocated in vitro across the peptidoglycancontaining cyanelle envelope. Efficient import was also observed in a heterologous system with pea chloroplasts as the recipient organelles. These results support the conclusion derived from comparative analysis of plastid genome organization, that all plastids originate from a common semi‐autonomous endosymbiotic ancestor.

Molecular Biology of Cyanelles

In a number of systematically unrelated eucaryotes, plastid-like organelles, termed cyanelles, are found which resemble present day cyanobacteria in morphology, biochemical organization of their photosynthetic apparatus, and in the presence of apeptidoglycan wall. The existence of cyanelles in species of different systematic position indicates repeated invasions ofheterotrophically living cells by cyanobacteria-like organisms in the evolutionary past. Only one of these cyanelle-bearing algae has been studied to some extent. Investigations into the genome and gene structure of the cyanelle of the unicellular eucaryotic alga Cyanophora paradoxa are reviewed. The cyanelle genome of approximately 130,000 bp includes more genes than the chromosomes of typical higher plant chloroplasts, mainly because only very few introns are found in cyanelle genes. Genes are tightly packed in operons that are similar in structure to bacterial operons. Up until now, with perhaps 85% of the cyanelle genome sequenced, genes that are typically found in chloroplasts are found in cyanelles as well. A remarkable exception is the apparent absence of ndh gene homologs. Other cyanelle genes, absent from chloroplasts, encode proteins functioning in isoprenoid biosynthesis, chlorophyll biosynthesis, other metabolic processes, and protein transport and protein folding. Generally, more of the genes encoding components of multi-protein complexes (ribosomes, photosystems, ATP synthase, etc.) are retained by cyanelles. In their gene complement cyanelles resemble chromophytic and rhodophytic algal plastids, while in their gene organization parallels exist to cyanobacteria as well as to higher plant plastids.

Partial characterization of the genome of the ‘endosymbiotic’ cyanelles from cyanophora paradoxa

Cyanophoru paradoxa, a flagellate of uncertain taxonomic position, is capable of growing photoautotrophically and its cyanelles are generally accepted to be endosymbiotic blue-green algae [ 11. In electron micrographs these ‘endosymbionts’ are characterized by a rudimentary cell wall which differentiates them clearly from any known type of chloroplast [2]. Furthermore the cyanelles have been shown to be sensitive to lysozyme [3] and to contain N-acetyl muramic acid and 2,6-diaminopimelic acid in their envelopes [4]. Unlike those from other host organisms the cyanelles from C’anophora paradoxa to date cannot be cultivated in vitro. It thus appears that these cyanelles represent semiautonomous organelles which could be envisaged as being intermediates between free living blue-green algae and chloroplasts [5]. The use of reassociation kinetics [6] to determine a genome size of 117 megadaltons for the DNA of these cyanelles seems to underline the above. This value is comparable to genome sizes of -90-l 30 megadaltons observed for ctDNAs of green algae and higher plants [7]. However the kinetic complexities from freeliving Cyanophyceae are in the range of 1.6-3 X lo9 daltons [8] and hence 1 order of magnitude larger. Here the isolation and purification of the cyanelle DNA and the results of its cleavage by restriction endonucleases are reported. We find a size 115 + 5 megadaltons and the results show in addition that the

Pathway of Cytosolic Starch Synthesis in the Model Glaucophyte Cyanophora paradoxa

ABSTRACT The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.

Cyanelle DNA from Cyanophora paradoxa

SummaryCyanelles which have been found in few eukaryotic organisms are photosynthetically active organelles which strikingly resemble cyanobacteria. The complexity of the cyanelle genome in Cyanophora paradoxa (127 Kbp) is too low to consider them as independent organisms in a symbiotic relationship. In order to correlate cyanelle genome and gene structure with those of plastid chromosomes of other plants, a circular map of the cyanelle DNA from Cyanophora paradoxa (strain LB555 UTEX) has been constructed using the restriction endonucleases SalI (generating 6 DNA fragments), BamHI (6), SalI (5), XhoI (9), and BglII (19).Besides the rRNA genes (16S, 23S, 5S), genes for 14 proteins have been located on this circular map. Among those are components of several multienzyme complexes involved in photosynthetic electron transport, as well as the large subunit of ribulose-1,5-bisphosphate carboxylase and two ribosomal proteins. All the probes used, were derived from a collection of spinach chloroplast DNA clones. Hybridization experiments showed signals to DNA fragments primarily from the large single-copy region of cyanelle DNA. The arrangement of genes on cyanelle DNA is different from that on spinach chloroplast DNA. However, genes which have been shown to be cotranscribed in spinach chloroplasts are also clustered on cyanelle DNA.

The cyanelle S10 spc ribosomal protein gene operon from Cyanophora paradoxa

SummaryIn Cyanophora paradoxa photosynthetic organelles termed cyanelles perform the functions of chloroplasts in higher plants, while the structural and biochemical characteristics of the cyanelle are essentially cyanobacterial. Our interest in studying the evolutionary relationship between cyanelles and chloroplasts led us to focus on cyanelle-encoded genes of the translational apparatus, specifically genes equivalent to those of the bacterial S10 and spc operons. The structure of a large ribosomal protein gene cluster from cyanelle DNA was characterized and compared with that from plastids and bacteria. Sequences of the following cyanelle genes encompassing 4.8 kb are reported here: 5′-rpl22-rps3-rpl16-rps17-rpl14-rpl5-rps8-rpl6-rpl18-rps5-3′. Cyanelles contain five more ribosomal protein genes than do higher plant chloroplasts and four more genes than Euglena gracilis plastids in the S10/spc region of this gene cluster. The gene encoding rpl36 is absent, in contrast to the case in other plastid DNAs. These genes, including the previously characterized genes rpl3, rpl2 and rps19, are transcribed as a primary transcript of ∼7500 nucleotides. The occurrence of transcripts smaller than this presumptive primary transcript suggests that it is processed into defined segments. Transcription terminates 3′ of rps5 where a 40 by hairpin with one mismatch (−42.2 kcal) may be folded. Immediately downstream of rps5 an open reading frame, ORF492, is contained on a separate transcript. A comparison of gene content, operon structure and deduced amino acid sequence of the genes in the S10 and spc operons from different organisms supports the notion that cyanelles are intermediary between known plastids and cyanobacteria.