The mitochondrial genome of Chlorogonium elongatum inferred from the complete sequence.

The 22,704-bp circular mitochondrial DNA (mtDNA) of the chlamydomonad alga Chlorogonium elongatum was completely cloned and sequenced. The genome encodes seven proteins of the respiratory electron transport chain, subunit 1 of the cytochrome oxidase complex (cox1), apocytochrome b (cob), five subunits of the NADH dehydrogenase complex (nad1, nad2, nad4, nad5, and nad6), a set of three tRNAs (Q, W, M), and the large (LSU)- and small (SSU)-subunit ribosomal RNAs. Six group-I introns were found, two each in the cox1, cob, and nad5 genes. In each intron an open reading frame (ORF) related to maturases or endonucleases was identified. Both the LSU and the SSU rRNA genes are split into fragments intermingled with each other and with other genes. Although the average A + T content is 62.2%, GC-rich clusters were detected in intergenic regions, in variable domains of the rRNA genes, and in introns and intron-encoded ORFs. A comparison of the genome maps reveals that C. elongatum and Chlamydomonas eugametos mtDNAs are more closely related to one another than either is to Chlamydomonas reinhardtii mtDNA.

The apocytochrome-b gene in Chlorogonium elongatum (Chlamydomonadaceae): an intronic GIY-YIG ORF in green algal mitochondria.

The mitochondrial cob gene from the green alga Chlorogonium elongatum (Chlamydomonadaceae) is interrupted by two group-I introns each containing an open reading frame in-phase with the upstream exon. One of these ORFs belongs to the LAGLI-DADG family, the other to the GIY-YIG family. The latter has not yet been identified in any mitochondrial genome except those from fungi. The Chlorogonium ORFs are similar to ORFs encoded by fungal introns that are located at an identical position within the gene, and to the ORF encoded by the mobile intron in the Chlamydomonas smithii cob gene.

Origin and evolution of mitochondria: what have we learnt from red algae?

The purpose of this review is to present an account of our current understanding of the structure, organization and evolution of mitochondrial genomes, and to discuss the origin and evolution of mitochondria from the perspective recently provided by the extensive sequencing of various mitochondrial genomes. Mitochondrial-en-coded protein phylogenies are congruent with nuclear phylogenies and strongly support a monophyletic origin of mitochondria. The newly available data from red-algal mitochondrial genomes, in particular, show that the structural and functional diversity of mitochondrial genomes can be accounted for by paralogous evolution. We also discuss the influence of other constraints, such as uniparental inheritance, on the evolution of genome organization in mitochondria.

The CMS-associated 16 kDa protein encoded by orfH522 in the PET1 cytoplasm is also present in other male-sterile cytoplasms of sunflower.

In sunflower plants carrying the PET1 cytoplasm male sterility (CMS) is associated with a new open reading frame (orfH522) in the 3'-flanking region of the atpA gene and an additional 16 kDa protein. Twenty-seven male-sterile cytoplasms of different origin were studied for the expression of the 16 kDa protein. In addition to the PET1 cytoplasm nine other male-sterile cytoplasms express the CMS-associated protein. These CMS sources originate from different interspecific crosses, from spontaneously occurring male-sterile plants in wild sunflower and from induced mutagenesis. Polyclonal antisera were raised against fusion proteins which contain 421 bp of the 3'-coding region of orfH522 to verify by immunological methods the identity of the other CMS cytoplasms. The anti-ORFH522 antiserum showed a positive reaction in the immunoblot with all CMS cytoplasms which expressing the 16 kDa protein. Investigations of the mitochondrial DNA demonstrated that all ten CMS cytoplasms which express the 16 kDa protein have the same organization at the atpA locus. OrfH522 as probes gave the same transcript pattern for the investigated CMS cytoplasms, just as for PET1. The MAX1 cytoplasm has an orfH522-related sequence but does not synthesize the 16 kDa protein. Using the sodium carbonate treatment the 16 kDa protein proved to be membrane-bound. Computer analyses predict that the hydrophobic N-terminal region of ORFH522 may form a transmembrane helix functioning as membrane anchor.

Genes for two subunits of succinate dehydrogenase form a cluster on the mitochondrial genome of Rhodophyta.

Mitochondrial DNA from the unicellular rhodophyte Cyanidium caldarium RK-1 and the multicellular Chondrus crispus were isolated, cloned, and sequenced. Two genes, sdhB and sdhC, that encode subunits of the succinate dehydrogenase, were identified by similarity. These genes form a cluster (sdhCB) in both red algae.

Physical map and gene organization of the mitochondrial genome from the unicellular green alga Platymonas (Tetraselmis) subcordiformis (Prasinophyceae).

The entire mitochondrial genome (mt genome) of the unicellular green alga Platymonas subcordiformis (synonym Tetraselmis subcordiformis; Prasinophyceae) was cloned and a physical map for the four restriction enzymes Hind III, Eco RI, Bg/II and Xba I was constructed. The mt genome of P. subcordiformis is a 42.8 kb circular molecule, coding for at least 23 genes. Hybridization and sequence analysis revealed the presence of a ca. 1.5 kb inverted repeat on the mt genome of P. subcordiformis. Phylogenetic analyses based on sequences of several coxI genes were carried out. Our data indicate that mitochondria from P. subcordiformis and from land plants form a natural, monophyletic group.

Opposite Regulation of the Copy Number and the Expression of Plastid and Mitochondrial Genes by Light and Acetate in the Green Flagellate Chlorogonium.

In the unicellular green alga Chlorogonium elongatum (Chlamydomonadaceae), the formation of both the photosynthetic and the respiratory apparatus is under the control of light and acetate. Autotrophically cultured cells possess a 3-fold higher copy number of the plastid genes rbcL and psbA than cells cultivated in the dark with acetate (heterotrophic cells). Under mixotrophic conditions (light and acetate), both genes are present at an intermediate level. This pattern is repeated at the mRNA level. The amounts of rbcL and psbA mRNAs are approximately 3-fold higher in autotrophic cells than in heterotrophic ones and are intermediate in mixotrophic cells. As expected, the copy number of the nuclear-encoded rbcS gene is constant irrespective of the applied culture conditions. RbcS mRNA, however, is 7-fold more frequent in autotrophic than in heterotrophic cells. Again, mixotrophic cells show an intermediate level. In contrast to genes encoding plastid proteins, the copy number and transcript level of the mitochondrial cob gene are approximately 5-fold higher in heterotrophic cells than in autotrophic ones. As before, mixotrophic cells take an intermediate position. Therefore, light and acetate control the genes involved in the formation of either the photosynthetic or the respiratory apparatus in a coordinated but opposite manner.

Functional loss of all ndh genes in an otherwise relatively unaltered plastid genome of the holoparasitic flowering plant Cuscuta reflexa.

We have cloned and sequenced an area of about 9.0 kb of the plastid DNA (ptDNA) from the holoparasitic flowering plant Cuscuta reflexa to investigate the evolutionary response of plastid genes to a reduced selective pressure. The region contains genes for the 16S rRNA, a subunit of a plastid NAD(P)H dehydrogenase (ndhB), three transfer RNAs (trnA, trnI, trnV) as well as the gene coding for the ribosomal protein S7 (rps7). While the other genes are strongly conserved in C. reflexa, the ndhB gene is a pseudogene due to many frameshift mutations. In addition we used heterologous gene probes to identify the other ndh genes encoded by the plastid genome in higher plants. No hybridization signals could be obtained, suggesting that these genes are either lost or strongly altered in the ptDNA of C. reflexa. Together with evidence of deleted genes in the ptDNA of C. reflexa, the plastid genome can be grouped into four classes reflecting a different evolutionary rate in each case. The phylogenetic position of Cuscuta and the significance of ndh genes in the plastid genome of higher plants are discussed.

Organization of plastid-encoded ATPase genes and flanking regions including homologues of infB and tsf in the thermophilic red alga Galdieria sulphuraria.

We have cloned and sequenced the plastid ATPase operons (atp1 and atp2) and flanking regions from the unicellular red alga Galdieria sulphuraria (Cyanidium caldarium). Six genes (5 atpI, H, G, F, D and A 3) are linked in atp1 encoding ATPase subunits a, c, b, b, delta and alpha, respectively. The atpF gene does not contain an intron and overlaps atpD by 1 bp. As in the genome of chloroplasts from land plants, the cluster is located downstream of rps2, but between this gene and atp1 we found the gene for the prokaryotic translation elongation factor TS. Downstream of atpA, we detected two open reading frames, one encoding a putative transport protein. The genes atpB and atpE, encoding ATPase subunits beta and epsilon, respectively, are linked in atp2, separated by a 2 bp spacer. Upstream of atpB, an uninterrupted orf167 was detected which is homologous to an intron-containing open reading frame in land plant chloroplasts. This orf167 is preceded on the opposite DNA strand by a homologue to initiation factor 2 in prokaryotes. The arrangement of atp1 and atp2 is the same as observed in the multicellular red alga Antithamnion sp., indicating a conserved genome arrangement in the red algal plastid genome. Differences compared to green chloroplast genomes suggest a large phylogenetic distance between red algae and green plants, while similarities in arrangement and sequence to chromophytic ATPase operons support a red algal origin of chlorophyll a/c-containing plastids or alternatively point to a common prokaryotic endosymbiont.

Glutamate synthase is plastid-encoded in a red alga: implications for the evolution of glutamate synthases.

An actively transcribed gene (glsF) encoding for ferredoxin-dependent glutamate synthase (Fd-GOGAT) was found on the plastid genome of the multicellular red alga Antithamnion sp. Fd-GOGAT is not plastid-encoded in chlorophytic plants, demonstrating that red algal plastid genomes encode for additional functions when compared to those known from green chloroplasts. Moreover, our results suggest that the plant Fd-GOGAT has an endosymbiotic origin. The same may not be true for NADPH-dependent GOGAT. In Antithamnion glsF is flanked upstream by cpcBA and downstream by psaC and is transcribed monocistronically. Implications of these results for the evolution of GOGAT enzymes and the plastid genome are discussed.

A large deletion in the plastid DNA of the holoparasitic flowering plant Cuscuta reflexa concerning two ribosomal proteins (rpl2, rpl23), one transfer RNA (trnI) and an ORF 2280 homologue.

We have determined the nucleotide sequence of a 5.3-kb region of the plastid DNA (ptDNA) from the heterotrophic holoparasitic plant Cuscuta reflexa. The cloned area contains genes for the D1-protein (32-kDa protein; psbA), tRNA(His) (trnH), ORF 740 (homologous to ORF 2280 from Nicotiana tabacum), ORF 77 (homologous to ORF 70), tRNA(Leu) (trnL) and a hypothetical ORF 55 which has no homology to any known gene among higher plants. This 5.3-kb area is colinear with a 12.4-kb region of tobacco ptDNA and has therefore undergone several deletions totalling 7.1 kb. Most of the missing nucleotides belong to one large deletion in the ptDNA of C. reflexa of approximately 6.5 kb. This deletion involves two ribosomal protein genes, rpl2 and rpl23, as well as the transfer RNA for Isoleucin (trnI) and a region encoding 1540 amino-acid residues of an ORF 2280 homologue, as compared to tobacco chloroplast DNA. This is remarkable since the remaining genes, especially the psbA gene, are highly conserved in C. reflexa. Furthermore, we found that the expression of the psbA gene is in the same range as in the autotrophic Ipomoea purpurea which belongs to the same family as Cuscuta (Convolvulaceae). Here we hypothesize a total loss of rpl2 and rpl23 in the entire genome of C. reflexa. The phylogenetic position of, and the evolutionary change of ptDNA from, Cuscuta are discussed.

Large ATP synthase operon of the red alga Antithamnion sp. resembles the corresponding operon in cyanobacteria.

The large plastid ATP synthase operon of the multicellular red alga Antithamnion sp. was cloned and the sequence of six ATPase genes determined. The operon resembles more the one from cyanobacteria than the ATP synthase operon of the chloroplast genome. The gene order is atpI, H, G, F, D and A, coding for the ATPase subunits a, c, b', b, delta and alpha, respectively. In green plants, the genes atpG and atpD are located in the nucleus. Unlike the situation in three published cyanobacterial ATP synthase operons, atpC, coding for the gamma subunit, is not a part of the rhodoplast operon. A single 4.5 kb transcript was detected with atpG, F, D and A gene probes that could span the whole operon, but no transcript could be detected with atpI and atpH probes. The end of an open reading frame preceding the atp genes shows remarkable homology to elongation factor TS from Escherichia coli. Behind the ATPase cluster, two open reading frames were detected that are not homologous to any known chloroplast gene. One of them may code for a transport protein of unknown specificity. Gene arrangement and sequence comparisons support the hypothesis of a polyphyletic origin of rhodoplasts and chloroplasts.

Organization and expression of a phycobiliprotein gene cluster from the unicellular red alga Cyanidium caldarium.

We have sequenced a plastid gene cluster from the unicellular red alga Cyanidium caldarium which is located downstream from the psbA gene and contains, in the following order, genes for a beta-allophycocyanin-like protein (apcB'), a putative 9.5 kDa allophycocyanin linker protein (apcL9.5) and a putative 29 kDa phycocyanin linker protein (cpcL29). The apcB' and apcL9.5 genes are organized in the form of an operon. The cpcL29 gene is transcribed monocistronically from the opposite strand of DNA. Both transcription units are probably terminated at a 25 bp inverted repeat 3 and 5 bp downstream of the stop codons of the apcL9.5 and cpcL29 genes, respectively. The levels of both transcripts are greatly reduced in the dark as is the psbA transcript. Downstream from the phycobiliprotein gene cluster two open reading frames (ORFs) were found which are homologous to ORFs from plastid DNAs and cyanelle DNA of Cyanophora paradoxa. Sequence homologies between genes analysed in this study and corresponding genes from cyanobacteria, chlorophytic plastids and cyanelles point to a large phylogenetic distance between the plastids of Cyanidium and cyanobacteria and other plastid types.

A 16 kb small single-copy region separates the plastid DNA inverted repeat of the unicellular red alga Cyanidium caldarium: physical mapping of the IR-flanking regions and nucleotide sequences of the psbD-psbC, rps16, 5S rRNA and rpl21 genes.

The four inverted repeat (IR) flanking regions of the Cyanidium caldarium plastid DNA were cloned. Southern blotting, transcript and sequence analyses of the border regions revealed the psbD-psbC operon and the rps16 gene within the large single-copy region upstream of the 16S rDNA gene and the rpl21 gene downstream of the 5S rDNA within the 16 kb small single-copy region. The size of the IR is ca. 5 kb. The nucleotide sequences of the psbD-psbC, rps16, rpl21 and 5S rRNA genes with the corresponding alignments and physical maps of the regions are presented. Northern analysis revealed a less complex psbD-psbC transcription pattern than has been found in higher plants. Comparisons to other red algal data point to structural diversity within red algal plastid DNA.

Structure and expression of a plastid-encoded groEL homologous heat-shock gene in a thermophilic unicellular red alga.

A gene homologous to the E. coli groEL locus was identified on the plastid genome of the unicellular red alga Cyanidium caldarium strain 14-1-1 (synonym: Galdieria sulphuraria). The complete nucleotide sequence was determined and compared to bacterial- and nuclear-encoded counterparts of higher plants. At the amino-acid level the C. caldarium gene shows 70% homology to the corresponding gene of the cyanobacterium Synechococcus and 52% homology to nuclear-encoded counterparts of higher plants, respectively. Northern and Western blot experiments were used to investigate the dependence of the transcript- and protein-level on culture temperature and heat shock.

Organization and sequence of photosynthetic genes from the plastid genome of the holoparasitic flowering plant Cuscuta reflexa.

We have cloned and sequenced an area of about 6 kb of the plastid DNA (ptDNA) from the holoparasitic plant Cuscuta reflexa. This region contains (in the following order) genes for the cytochrome b6 f-complex subunit V (petG), tRNA(Val) (trnV), tRNA(Met) (trnM), the epsilon- and beta-subunit of the chloroplast ATP-synthase (atpE and atpB) and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; rbcL). In addition we identified other photosynthesis-related genes (atpA, petB, psaA, psbA, psbB, psbC, and psbD) in C. reflexa by heterologous hybridization. The gene arrangement of the sequenced area is, except for the petG gene, the same as in ptDNAs of other higher plants (e.g. Nicotiana tabacum). Sequence homologies between the Cuscuta genes and corresponding genes from higher plants are in the range of 90%. The only significant difference is that the rbcL gene of C. reflexa encodes a polypeptide which is 18-23 amino acids longer than in other higher plants. This is remarkable since C. reflexa has lost its ability to grow photoautotrophically. The transcript level of the rbcL gene, however, is strongly reduced as compared to tobacco. These findings are compatible with results from Western blotting analysis, where no Rubisco large subunit was detectable, and with the lack of Rubisco activity in crude extracts of C. reflexa.

Structural similarities between psbA genes from red and brown algae.

The single copy psbA genes from the multicellular red alga Antithamnion spec. and the brown alga Ectocarpus siliculosus have been cloned and sequenced and monocistronic transcripts have been detected. Both genes contain an insertion of 21 bp at the 3' end which was also found in cyanobacteria and which is absent in chloroplasts and the chlorophyll b-containing prochlorophyte Prochlorothrix hollandica. These findings are in agreement with the hypothesis of a polyphyletic origin of plastids. Plastids of red and brown algae appear to be closely related.

Cytoplasmic male sterility in sunflower is correlated with the co-transcription of a new open reading frame with the atpA gene.

The organization and expression of the mitochondrial (mt) genome of fertile, male-sterile and restored lines of Helianthus annuus and of H. petiolaris were compared to identify alterations which might lead to cytoplasmic male sterility (CMS). The mtDNAs of fertile and male-sterile lines differ by an 11 kb inversion and a 5 kb insertion. The rearrangements seem to be the result of recombination events within an inverted repeat of 261 bp. Detectable alterations in the transcript pattern of the rearranged mtDNA regions are restricted to the atpA locus. The male-sterile line CMSBaso shows three additional transcripts of the atpA locus of about 2500, 1200 and 250 nucleotides which are not detectable in Baso. However, the coding sequences of the atpA gene are entirely identical in the fertile line Baso and the male-sterile line CMSBaso. But a new open reading frame (orfH522) of 522 nucleotides is co-transcribed with the atpA gene as an additional larger transcript of about 2500 nucleotides in CMSBaso. orfH522 is also included in a second additional transcript of about 1200 nucleotides. The predicted translation product of orfH522 might play a role in CMS in sunflower.