Claude Lemieux

Ancestral chloroplast genome in Mesostigma viride reveals an early branch of green plant evolution.

Sequence comparisons suggest that all living green plants belong to one of two major phyla: Streptophyta (land plants and their closest green algal relatives, the charophytes); and Chlorophyta (the rest of green algae). Because no green algae are known that pre-date the Streptophyta/Chlorophyta split, and also because the earliest diverging green algae show considerable morphological variation, the nature of the unicellular flagellate ancestor of the two green plant phyla is unknown. Here we report that the flagellate Mesostigma viride belongs to the earliest diverging green plant lineage discovered to date. We have sequenced the entire chloroplast DNA (118,360 base pairs) of this green alga and have conducted phylogenetic analyses of sequences derived from this genome. Mesostigma represents a lineage that emerged before the divergence of the Streptophyta and Chlorophyta, a position that is supported by several features of its chloroplast DNA. The structure and gene organization of this genome indicate that chloroplast DNA architecture has been extremely well conserved in the line leading to land plants.

The chloroplast ycf3 and ycf4 open reading frames of Chlamydomonas reinhardtii are required for the accumulation of the photosystem I complex

The chloroplast genes ycf3 and ycf4 from the green alga Chlamydomonas reinhardtii have been characterized. The deduced amino acid sequences of Ycf4 (197 residues) and Ycf3 (172 residues) display 41–52% and 64–78% sequence identity, respectively, with their homologues from algae, land plants and cyanobacteria. In C.reinhardtii, ycf4 and ycf3 are co‐transcribed as members of the rps9–ycf4–ycf3–rps18 polycistronic transcriptional unit into RNAs of 8.0 kb and 3.0 kb corresponding to the entire unit and to rps9–ycf4–ycf3, respectively. Using biolistic transformation, ycf4 and ycf3 were disrupted with a chloroplast selectable marker cassette. Transformants lacking ycf4 or ycf3 were unable to grow photoautotrophically and were deficient in photosystem I activity. Western blot analysis showed that the photosystem I (PSI) complex does not accumulate stably in thylakoid membranes of these transformants. Ycf4 and Ycf3 were localized on thylakoid membranes but not stably associated with the PSI complex and accumulated to wild‐type levels in mutants lacking PSI. RNA blot hybridizations showed that transcripts of psaA, psaB and psaC accumulate normally in these mutants and use of chimeric reporter genes revealed that Ycf3 is not required for initiation of translation of psaA and psaB mRNA. Our results indicate that Ycf3 and Ycf4 are required for stable accumulation of the PSI complex.

The chloroplast and mitochondrial genome sequences of the charophyte Chaetosphaeridium globosum: Insights into the timing of the events that restructured organelle DNAs within the green algal lineage that led to land plants

The land plants and their immediate green algal ancestors, the charophytes, form the Streptophyta. There is evidence that both the chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) underwent substantial changes in their architecture (intron insertions, gene losses, scrambling in gene order, and genome expansion in the case of mtDNA) during the evolution of streptophytes; however, because no charophyte organelle DNAs have been sequenced completely thus far, the suite of events that shaped streptophyte organelle genomes remains largely unknown. Here, we have determined the complete cpDNA (131,183 bp) and mtDNA (56,574 bp) sequences of the charophyte Chaetosphaeridium globosum (Coleochaetales). At the levels of gene content (124 genes), intron composition (18 introns), and gene order, Chaetosphaeridium cpDNA is remarkably similar to land-plant cpDNAs, implying that most of the features characteristic of land-plant lineages were gained during the evolution of charophytes. Although the gene content of Chaetosphaeridium mtDNA (67 genes) closely resembles that of the bryophyte Marchantia polymorpha (69 genes), this charophyte mtDNA differs substantially from its land-plant relatives at the levels of size, intron composition (11 introns), and gene order. Our finding that it shares only one intron with its land-plant counterparts supports the idea that the vast majority of mitochondrial introns in land plants appeared after the emergence of these organisms. Our results also suggest that the events accounting for the spacious intergenic spacers found in land-plant mtDNAs took place late during the evolution of charophytes or coincided with the transition from charophytes to land plants.

The Complete Mitochondrial DNA Sequences of Nephroselmis olivacea and Pedinomonas minor : Two Radically Different Evolutionary Patterns within Green Algae

Green plants appear to comprise two sister lineages, Chlorophyta (classes Chlorophyceae, Ulvophyceae, Trebouxiophyceae, and Prasinophyceae) and Streptophyta (Charophyceae and Embryophyta, or land plants). To gain insight into the nature of the ancestral green plant mitochondrial genome, we have sequenced the mitochondrial DNAs (mtDNAs) of Nephroselmis olivacea and Pedinomonas minor. These two green algae are presumptive members of the Prasinophyceae. This class is thought to include descendants of the earliest diverging green algae. We find that Nephroselmis and Pedinomonas mtDNAs differ markedly in size, gene content, and gene organization. Of the green algal mtDNAs sequenced so far, that of Nephroselmis (45,223 bp) is the most ancestral (minimally diverged) and occupies the phylogenetically most basal position within the Chlorophyta. Its repertoire of 69 genes closely resembles that in the mtDNA of Prototheca wickerhamii, a later diverging trebouxiophycean green alga. Three of the Nephroselmis genes (nad10, rpl14, and rnpB) have not been identified in previously sequenced mtDNAs of green algae and land plants. In contrast, the 25,137-bp Pedinomonas mtDNA contains only 22 genes and retains few recognizably ancestral features. In several respects, including gene content and rate of sequence divergence, Pedinomonas mtDNA resembles the reduced mtDNAs of chlamydomonad algae, with which it is robustly affiliated in phylogenetic analyses. Our results confirm the existence of two radically different patterns of mitochondrial genome evolution within the green algae.

The Chloroplast Genomes of the Green Algae Pyramimonas, Monomastix, and Pycnococcus Shed New light on the Evolutionary History of Prasinophytes and the Origin of the Secondary Chloroplasts of Euglenids

Because they represent the earliest divergences of the Chlorophyta and include the smallest known eukaryotes (e.g., the coccoid Ostreococcus), the morphologically diverse unicellular green algae making up the Prasinophyceae are central to our understanding of the evolutionary patterns that accompanied the radiation of chlorophytes and the reduction of cell size in some lineages. Seven prasinophyte lineages, four of which exhibit a coccoid cell organization (no flagella nor scales), were uncovered from analysis of nuclear-encoded 18S rDNA data; however, their order of divergence remains unknown. In this study, the chloroplast genome sequences of the scaly quadriflagellate Pyramimonas parkeae (clade I), the coccoid Pycnococcus provasolii (clade V), and the scaly uniflagellate Monomastix (unknown affiliation) were determined, annotated, and compared with those previously reported for green algae/land plants, including two prasinophytes (Nephroselmis olivacea, clade III and Ostreococcus tauri, clade II). The chlorarachniophyte Bigelowiella natans and the euglenid Euglena gracilis, whose chloroplasts originate presumably from distinct green algal endosymbionts, were also included in our comparisons. The three newly sequenced prasinophyte genomes differ considerably from one another and from their homologs in overall structure, gene content, and gene order, with the 80,211-bp Pycnococcus and 114,528-bp Monomastix genomes (98 and 94 conserved genes, respectively) resembling the 71,666-bp Ostreococcus genome (88 genes) in featuring a significantly reduced gene content. The 101,605-bp Pyramimonas genome (110 genes) features two conserved genes (rpl22 and ycf65) and ancestral gene linkages previously unrecognized in chlorophytes as well as a DNA primase gene putatively acquired from a virus. The Pyramimonas and Euglena cpDNAs revealed uniquely shared derived gene clusters. Besides providing unequivocal evidence that the green algal ancestor of the euglenid chloroplasts belonged to the Pyramimonadales, phylogenetic analyses of concatenated chloroplast genes and proteins elucidated the position of Monomastix and showed that the Mamiellales, a clade comprising Ostreococcus and Monomastix, are sister to the Pyramimonadales + Euglena clade. Our results also revealed that major reduction in gene content and restructuring of the chloroplast genome occurred in conjunction with important changes in cell organization in at least two independent prasinophyte lineages, the Mamiellales and the Pycnococcaceae.

Flexible DNA Target Site Recognition by Divergent Homing Endonuclease Isoschizomers I-CreI and I-MsoI

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.

The Mitochondrial Genome of Chara vulgaris: Insights into the Mitochondrial DNA Architecture of the Last Common Ancestor of Green Algae and Land Plants

Mitochondrial DNA (mtDNA) has undergone radical changes during the evolution of green plants, yet little is known about the dynamics of mtDNA evolution in this phylum. Land plant mtDNAs differ from the few green algal mtDNAs that have been analyzed to date by their expanded size, long spacers, and diversity of introns. We have determined the mtDNA sequence of Chara vulgaris (Charophyceae), a green alga belonging to the charophycean order (Charales) that is thought to be the most closely related alga to land plants. This 67,737-bp mtDNA sequence, displaying 68 conserved genes and 27 introns, was compared with those of three angiosperms, the bryophyte Marchantia polymorpha, the charophycean alga Chaetosphaeridium globosum (Coleochaetales), and the green alga Mesostigma viride. Despite important differences in size and intron composition, Chara mtDNA strikingly resembles Marchantia mtDNA; for instance, all except 9 of 68 conserved genes lie within blocks of colinear sequences. Overall, our genome comparisons and phylogenetic analyses provide unequivocal support for a sister-group relationship between the Charales and the land plants. Only four introns in land plant mtDNAs appear to have been inherited vertically from a charalean algar ancestor. We infer that the common ancestor of green algae and land plants harbored a tightly packed, gene-rich, and relatively intron-poor mitochondrial genome. The group II introns in this ancestral genome appear to have spread to new mtDNA sites during the evolution of bryophytes and charalean green algae, accounting for part of the intron diversity found in Chara and land plant mitochondria.

A clade uniting the green algae Mesostigma viride and Chlorokybus atmophyticus represents the deepest branch of the Streptophyta in chloroplast genome-based phylogenies.

BackgroundThe Viridiplantae comprise two major phyla: the Streptophyta, containing the charophycean green algae and all land plants, and the Chlorophyta, containing the remaining green algae. Despite recent progress in unravelling phylogenetic relationships among major green plant lineages, problematic nodes still remain in the green tree of life. One of the major issues concerns the scaly biflagellate Mesostigma viride, which is either regarded as representing the earliest divergence of the Streptophyta or a separate lineage that diverged before the Chlorophyta and Streptophyta. Phylogenies based on chloroplast and mitochondrial genomes support the latter view. Because some green plant lineages are not represented in these phylogenies, sparse taxon sampling has been suspected to yield misleading topologies. Here, we describe the complete chloroplast DNA (cpDNA) sequence of the early-diverging charophycean alga Chlorokybus atmophyticus and present chloroplast genome-based phylogenies with an expanded taxon sampling.ResultsThe 152,254 bp Chlorokybus cpDNA closely resembles its Mesostigma homologue at the gene content and gene order levels. Using various methods of phylogenetic inference, we analyzed amino acid and nucleotide data sets that were derived from 45 protein-coding genes common to the cpDNAs of 37 green algal/land plant taxa and eight non-green algae. Unexpectedly, all best trees recovered a robust clade uniting Chlorokybus and Mesostigma. In protein trees, this clade was sister to all streptophytes and chlorophytes and this placement received moderate support. In contrast, gene trees provided unequivocal support to the notion that the Mesostigma + Chlorokybus clade represents the earliest-diverging branch of the Streptophyta. Independent analyses of structural data (gene content and/or gene order) and of subsets of amino acid data progressively enriched in slow-evolving sites led us to conclude that the latter topology reflects the true organismal relationships.ConclusionIn disclosing a sister relationship between the Mesostigmatales and Chlorokybales, our study resolves the long-standing debate about the nature of the unicellular flagellated ancestors of land plants and alters significantly our concepts regarding the evolution of streptophyte algae. Moreover, in predicting a richer chloroplast gene repertoire than previously inferred for the common ancestor of all streptophytes, our study has contributed to a better understanding of chloroplast genome evolution in the Viridiplantae.

Mobile introns: definition of terms and recommended nomenclature

A number of introns in mitochondrial, chloroplast, nuclear or prokaryotic genes have recently been shown to encode double-strand sequence-specific endonucleases. Such introns are mobile genetic elements that insert themselves at or near the cleaved sites. A uniform nomenclature to designate the molecular elements involved in the phenomenon of intron mobility is proposed.

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth.

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wildtype cells under phototrophic conditions.