Charophyte Green Algae Research Articles

Transcriptome Profiling of the Green Alga Spirogyra pratensis (Charophyta) Suggests an Ancestral Role for Ethylene in Cell Wall Metabolism, Photosynthesis, and Abiotic Stress Responses.

It is well known that ethylene regulates a diverse set of developmental and stress-related processes in angiosperms, yet its roles in early-diverging embryophytes and algae are poorly understood. Recently, it was shown that ethylene functions as a hormone in the charophyte green alga Spirogyra pratensis Since land plants evolved from charophytes, this implies conservation of ethylene as a hormone in green plants for at least 450 million years. However, the physiological role of ethylene in charophyte algae has remained unknown. To gain insight into ethylene responses in Spirogyra, we used mRNA sequencing to measure changes in gene expression over time in Spirogyra filaments in response to an ethylene treatment. Our analyses show that at the transcriptional level, ethylene predominantly regulates three processes in Spirogyra: (1) modification of the cell wall matrix by expansins and xyloglucan endotransglucosylases/hydrolases, (2) down-regulation of chlorophyll biosynthesis and photosynthesis, and (3) activation of abiotic stress responses. We confirmed that the photosynthetic capacity and chlorophyll content were reduced by an ethylene treatment and that several abiotic stress conditions could stimulate cell elongation in an ethylene-dependent manner. We also found that the Spirogyra transcriptome harbors only 10 ethylene-responsive transcription factor (ERF) homologs, several of which are regulated by ethylene. These results provide an initial understanding of the hormonal responses induced by ethylene in Spirogyra and help to reconstruct the role of ethylene in ancestral charophytes prior to the origin of land plants.

Hyperspectral imaging of snow algae and green algae from aeroterrestrial habitats

Snow algae and green algae living in aeroterrestrial habitats are ideal objects to study adaptation to high light irradiation. Here, we used a detailed description of the spectral properties as a proxy for photo-acclimation/protection in snow algae (Chlamydomonas nivalis, Chlainomonas sp. and Chloromonas sp.) and charophyte green algae (Zygnema sp., Zygogonium ericetorum and Klebsormidium crenulatum). The hyperspectral microscopic mapping and imaging technique allowed us to acquire total absorption spectra of these microalgae in the waveband of 400–900nm. Particularly in Chlamydomonas nivalis and Chlainomonas sp., a high absorbance between 400–550nm was observed, due to naturally occurring secondary carotenoids; in Chloromonas sp. and in the charopyhte algae this high absorbance was missing, the latter being close relatives to land plants. To investigate if cellular water loss has an influence on the spectral properties, the cells were plasmolysed in sorbitol or desiccated at ambient air. While in snow algae, these treatments did hardly change the spectral properties, in the charopyhte algae the condensation of the cytoplasm and plastids increased the absorbance in the lower waveband of 400–500nm. These changes might be ecologically relevant and photoprotective, as aeroterrestrial algae are naturally exposed to occasional water limitation, leading to desiccation, which are conditions usually occurring together with higher irradiation.

Abiotic Stress Tolerance of Charophyte Green Algae: New Challenges for Omics Techniques.

Charophyte green algae are a paraphyletic group of freshwater and terrestrial green algae, comprising the classes of Chlorokybophyceae, Coleochaetophyceae, Klebsormidiophyceae, Zygnematophyceae, Mesostigmatophyceae, and Charo- phyceae. Zygnematophyceae (Conjugating green algae) are considered to be closest algal relatives to land plants (Embryophyta). Therefore, they are ideal model organisms for studying stress tolerance mechanisms connected with transition to land, one of the most important events in plant evolution and the Earth’s history. In Zygnematophyceae, but also in Coleochaetophyceae, Chlorokybophyceae, and Klebsormidiophyceae terrestrial members are found which are frequently exposed to naturally occurring abiotic stress scenarios like desiccation, freezing and high photosynthetic active (PAR) as well as ultraviolet (UV) irradiation. Here, we summarize current knowledge about various stress tolerance mechanisms including insight provided by pioneer transcriptomic and proteomic studies. While formation of dormant spores is a typical strategy of freshwater classes, true terrestrial groups are stress tolerant in vegetative state. Aggregation of cells, flexible cell walls, mucilage production and accumulation of osmotically active compounds are the most common desiccation tolerance strategies. In addition, high photophysiological plasticity and accumulation of UV-screening compounds are important protective mechanisms in conditions with high irradiation. Now a shift from classical chemical analysis to next-generation genome sequencing, gene reconstruction and annotation, genome-scale molecular analysis using omics technologies followed by computer-assisted analysis will give new insights in a systems biology approach. For example, changes in transcriptome and role of phytohormone signaling in Klebsormidium during desiccation were recently described. Application of these modern approaches will deeply enhance our understanding of stress reactions in an unbiased non-targeted view in an evolutionary context.

Ethylene and Hormonal Cross Talk in Vegetative Growth and Development.

Ethylene is a gaseous plant hormone that most likely became a functional hormone during the evolution of charophyte green algae, prior to land colonization. From this ancient origin, ethylene evolved into an important growth regulator that is essential for myriad plant developmental processes. In vegetative growth, ethylene appears to have a dual role, stimulating and inhibiting growth, depending on the species, tissue, and cell type, developmental stage, hormonal status, and environmental conditions. Moreover, ethylene signaling and response are part of an intricate network in cross talk with internal and external cues. Besides being a crucial factor in the growth control of roots and shoots, ethylene can promote flowering, fruit ripening and abscission, as well as leaf and petal senescence and abscission and, hence, plays a role in virtually every phase of plant life. Last but not least, together with jasmonates, salicylate, and abscisic acid, ethylene is important in steering stress responses.

The evolution of WRKY transcription factors.

BackgroundThe availability of increasing numbers of sequenced genomes has necessitated a re-evaluation of the evolution of the WRKY transcription factor family. Modern day plants descended from a charophyte green alga that colonized the land between 430 and 470 million years ago. The first charophyte genome sequence from Klebsormidium flaccidum filled a gap in the available genome sequences in the plant kingdom between unicellular green algae that typically have 1-3 WRKY genes and mosses that contain 30-40. WRKY genes have been previously found in non-plant species but their occurrence has been difficult to explain.ResultsOnly two WRKY genes are present in the Klebsormidium flaccidum genome and the presence of a Group IIb gene was unexpected because it had previously been thought that Group IIb WRKY genes first appeared in mosses. We found WRKY transcription factor genes outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa. This patchy distribution suggests that lateral gene transfer is responsible. These lateral gene transfer events appear to pre-date the formation of the WRKY groups in flowering plants. Flowering plants contain proteins with domains typical for both resistance (R) proteins and WRKY transcription factors. R protein-WRKY genes have evolved numerous times in flowering plants, each type being restricted to specific flowering plant lineages. These chimeric proteins contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. Once formed, R protein WRKY genes may combine different components of signalling pathways that may either create new diversity in signalling or accelerate signalling by short circuiting signalling pathways.ConclusionsWe propose that the evolution of WRKY transcription factors includes early lateral gene transfers to non-plant organisms and the occurrence of algal WRKY genes that have no counterparts in flowering plants. We propose two alternative hypotheses of WRKY gene evolution: The “Group I Hypothesis” sees all WRKY genes evolving from Group I C-terminal WRKY domains. The alternative “IIa + b Separate Hypothesis” sees Groups IIa and IIb evolving directly from a single domain algal gene separate from the Group I-derived lineage.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0456-y) contains supplementary material, which is available to authorized users.

Conservation of ethylene as a plant hormone over 450 million years of evolution.

Land plants evolved more than 450 million years ago from a lineage of freshwater charophyte green algae(1). The extent to which plant signalling systems existed before the evolutionary transition to land is unknown. Although charophytes occupy a key phylogenetic position for elucidating the origins of such signalling systems(2-4), there is a paucity of sequence data for these organisms(5,6). Here we carry out de novo transcriptomics of five representative charophyte species, and find putative homologues for the biosynthesis, transport, perception and signalling of major plant hormones. Focusing on the plant hormone ethylene, we provide evidence that the filamentous charophyte Spirogyra pratensis possesses an ethylene hormone system homologous to that in plants. Spirogyra produces ethylene and exhibits a cell elongation response to ethylene. Spirogyra ethylene-signalling homologues partially rescue mutants of the angiosperm Arabidopsis thaliana and respond post-translationally to ethylene when expressed in plant cells, indicative of unambiguously homologous ethylene-signalling pathways in Spirogyra and Arabidopsis. These findings imply that the common aquatic ancestor possessed this pathway prior to the colonization of land and that cell elongation was possibly an ancestral ethylene response. This highlights the importance of charophytes for investigating the origins of fundamental plant processes.

Evidence for land plant cell wall biosynthetic mechanisms in charophyte green algae.

The charophyte green algae (CGA) are thought to be the closest living relatives to the land plants, and ancestral CGA were unique in giving rise to the land plant lineage. The cell wall has been suggested to be a defining structure that enabled the green algal ancestor to colonize land. These cell walls provide support and protection, are a source of signalling molecules, and provide developmental cues for cell differentiation and elongation. The cell wall of land plants is a highly complex fibre composite, characterized by cellulose cross-linked by non-cellulosic polysaccharides, such as xyloglucan, embedded in a matrix of pectic polysaccharides. How the land plant cell wall evolved is currently unknown: early-divergent chlorophyte and prasinophyte algae genomes contain a low number of glycosyl transferases (GTs), while land plants contain hundreds. The number of GTs in CGA is currently unknown, as no genomes are available, so this study sought to give insight into the evolution of the biosynthetic machinery of CGA through an analysis of available transcriptomes. Available CGA transcriptomes were mined for cell wall biosynthesis GTs and compared with GTs characterized in land plants. In addition, gene cloning was employed in two cases to answer important evolutionary questions. Genetic evidence was obtained indicating that many of the most important core cell wall polysaccharides have their evolutionary origins in the CGA, including cellulose, mannan, xyloglucan, xylan and pectin, as well as arabino-galactan protein. Moreover, two putative cellulose synthase-like D family genes (CSLDs) from the CGA species Coleochaete orbicularis and a fragment of a putative CSLA/K-like sequence from a CGA Spirogyra species were cloned, providing the first evidence that all the cellulose synthase/-like genes present in early-divergent land plants were already present in CGA. The results provide new insights into the evolution of cell walls and support the notion that the CGA were pre-adapted to life on land by virtue of the their cell wall biosynthetic capacity. These findings are highly significant for understanding plant cell wall evolution as they imply that some features of land plant cell walls evolved prior to the transition to land, rather than having evolved as a result of selection pressures inherent in this transition.

Comparative phylogenomics of the CBL-CIPK calcium-decoding network in the moss Physcomitrella, Arabidopsis, and other green lineages.

Land plants have evolved a host of anatomical and molecular adaptations for terrestrial growth. Many of these adaptations are believed to be elaborations of features that were present in their algal-like progenitors. In the model plant Arabidopsis, 10 Calcineurin B-Like proteins (CBLs) function as calcium sensors and modulate the activity of 26 CBL-Interacting Protein Kinases (CIPKs). The CBL-CIPK network coordinates environmental responses and helps maintain proper ion balances, especially during abiotic stress. We identified and analyzed CBL and CIPK homologs in green lineages, including CBLs and CIPKs from charophyte green algae, the closest living relatives of land plants. Phylogenomic evidence suggests that the network expanded from a small module, likely a single CBL-CIPK pair, present in the ancestor of modern plants and algae. Extreme conservation of the NAF motif, which mediates CBL-CIPK physical interactions, among all identified CIPKs supports the interpretation of CBL and CIPK homologs in green algae and early diverging land plants as functionally linked network components. We identified the full complement of CBL and CIPK loci in the genome of Physcomitrella, a model moss. These analyses demonstrate the strong effects of a recent moss whole genome duplication: CBL and CIPK loci appear in cognate pairs, some of which appear to be pseudogenes, with high sequence similarity. We cloned all full-length transcripts from these loci and performed yeast two-hybrid analyses to demonstrate CBL-CIPK interactions and identify specific connections within the network. Using phylogenomics, we have identified three ancient types of CBLs that are discernible by N-terminal localization motifs and a “green algal-type” clade of CIPKs with members from Physcomitrella and Arabidopsis.

Pectin Metabolism and Assembly in the Cell Wall of the Charophyte Green Alga Penium margaritaceum

The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.

Stable transformation and reverse genetic analysis of Penium margaritaceum: a platform for studies of charophyte green algae, the immediate ancestors of land plants

The charophyte green algae (CGA, Streptophyta, Viridiplantae) occupy a key phylogenetic position as the immediate ancestors of land plants but, paradoxically, are less well-studied than the other major plant lineages. This is particularly true in the context of functional genomic studies, where the lack of an efficient protocol for their stable genetic transformation has been a major obstacle. Observations of extant CGA species suggest the existence of some of the evolutionary adaptations that had to occur for land colonization; however, to date, there has been no robust experimental platform to address this genetically. We present a protocol for high-throughput Agrobacterium tumefaciens-mediated transformation of Penium margaritaceum, a unicellular CGA species. The versatility of Penium as a model for studying various aspects of plant cell biology and development was illustrated through non-invasive visualization of protein localization and dynamics in living cells. In addition, the utility of RNA interference (RNAi) for reverse genetic studies was demonstrated by targeting genes associated with cell wall modification (pectin methylesterase) and biosynthesis (cellulose synthase). This provided evidence supporting current models of cell wall assembly and inter-polymer interactions that were based on studies of land plants, but in this case using direct observation in vivo. This new functional genomics platform has broad potential applications, including studies of plant organismal biology and the evolutionary innovations required for transition from aquatic to terrestrial habitats.

Characterization of the Heterotrimeric G-Protein Complex and Its Regulator from the Green Alga Chara braunii Expands the Evolutionary Breadth of Plant G-Protein Signaling

The lack of heterotrimeric G-protein homologs in the sequenced genomes of green algae has led to the hypothesis that, in plants, this signaling mechanism coevolved with the embryophytic life cycle and the acquisition of terrestrial habitat. Given the large evolutionary gap that exists between the chlorophyte green algae and most basal land plants, the bryophytes, we evaluated the presence of this signaling complex in a charophyte green alga, Chara braunii, proposed to be the closest living relative of land plants. The C. braunii genome encodes for the entire G-protein complex, the Gα, Gβ, and Gγ subunits, and the REGULATOR OF G-PROTEIN SIGNALING (RGS) protein. The biochemical properties of these proteins and their cross-species functionality show that they are functional homologs of canonical G-proteins. The subunit-specific interactions between CbGα and CbGβ, CbGβ and CbGγ, and CbGα and CbRGS are also conserved, establishing the existence of functional G-protein complex-based signaling mechanisms in green algae.

Evolution of the plant–microbe symbiotic ‘toolkit’

Beneficial associations between plants and arbuscular mycorrhizal fungi play a major role in terrestrial environments and in the sustainability of agroecosystems. Proteins, microRNAs, and small molecules have been identified in model angiosperms as required for the establishment of arbuscular mycorrhizal associations and define a symbiotic 'toolkit' used for other interactions such as the rhizobia-legume symbiosis. Based on recent studies, we propose an evolutionary framework for this toolkit. Some components appeared recently in angiosperms, whereas others are highly conserved even in land plants unable to form arbuscular mycorrhizal associations. The exciting finding that some components pre-date the appearance of arbuscular mycorrhizal fungi suggests the existence of unknown roles for this toolkit and even the possibility of symbiotic associations in charophyte green algae.

Occurrence of homospermidine and thermospermine as a cellular polyamine in unicellular chlorophyte and multicellular charophyte green algae

Occurrence of homospermidine and thermospermine as a cellular polyamine in unicellular chlorophyte and multicellular charophyte green algae

Strigolactones affect development in primitive plants. The missing link between plants and arbuscular mycorrhizal fungi?

Strigolactones affect development in primitive plants. The missing link between plants and arbuscular mycorrhizal fungi?

Broad Phylogenomic Sampling and the Sister Lineage of Land Plants

The tremendous diversity of land plants all descended from a single charophyte green alga that colonized the land somewhere between 430 and 470 million years ago. Six orders of charophyte green algae, in addition to embryophytes, comprise the Streptophyta s.l. Previous studies have focused on reconstructing the phylogeny of organisms tied to this key colonization event, but wildly conflicting results have sparked a contentious debate over which lineage gave rise to land plants. The dominant view has been that ‘stoneworts,’ or Charales, are the sister lineage, but an alternative hypothesis supports the Zygnematales (often referred to as “pond scum”) as the sister lineage. In this paper, we provide a well-supported, 160-nuclear-gene phylogenomic analysis supporting the Zygnematales as the closest living relative to land plants. Our study makes two key contributions to the field: 1) the use of an unbiased method to collect a large set of orthologs from deeply diverging species and 2) the use of these data in determining the sister lineage to land plants. We anticipate this updated phylogeny not only will hugely impact lesson plans in introductory biology courses, but also will provide a solid phylogenetic tree for future green-lineage research, whether it be related to plants or green algae.

Phylogeny and Molecular Evolution of the Green Algae

The green lineage (Viridiplantae) comprises the green algae and their descendants the land plants, and is one of the major groups of oxygenic photosynthetic eukaryotes. Current hypotheses posit the early divergence of two discrete clades from an ancestral green flagellate. One clade, the Chlorophyta, comprises the early diverging prasinophytes, which gave rise to the core chlorophytes. The other clade, the Streptophyta, includes the charophyte green algae from which the land plants evolved. Multi-marker and genome scale phylogenetic studies have greatly improved our understanding of broad-scale relationships of the green lineage, yet many questions persist, including the branching orders of the prasinophyte lineages, the relationships among core chlorophyte clades (Chlorodendrophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae), and the relationships among the streptophytes. Current phylogenetic hypotheses provide an evolutionary framework for molecular evolutionary studies and comparative genomics. This review summarizes our current understanding of organelle genome evolution in the green algae, genomic insights into the ecology of oceanic picoplanktonic prasinophytes, molecular mechanisms underlying the evolution of complexity in volvocine green algae, and the evolution of genetic codes and the translational apparatus in green seaweeds. Finally, we discuss molecular evolution in the streptophyte lineage, emphasizing the genetic facilitation of land plant origins.

Uncovering the evolutionary origin of plant molecular processes: comparison of Coleochaete (Coleochaetales) and Spirogyra (Zygnematales) transcriptomes

BackgroundThe large and diverse land plant lineage is nested within a clade of fresh water green algae, the charophytes. Collection of genome-scale data for land plants and other organisms over the past decade has invigorated the field of evolutionary biology. One of the core questions in the field asks: how did a colonization event by a green algae over 450 mya lead to one of the most successful lineages on the tree of life? This question can best be answered using the comparative method, the first step of which is to gather genome-scale data across closely related lineages to land plants. Before sequencing an entire genome it is useful to first gather transcriptome data: it is less expensive, it targets the protein coding regions of the genome, and provides support for gene models for future genome sequencing. We built Expressed Sequence Tag (EST) libraries for two charophyte species, Coleochaete orbicularis (Coleochaetales) and Spirogyra pratensis (Zygnematales). We used both Sanger sequencing and next generation 454 sequencing to cover as much of the transcriptome as possible.ResultsOur sequencing effort for Spirogyra pratensis yielded 9,984 5' Sanger reads plus 598,460 GS FLX Standard 454 sequences; Coleochaete orbicularis yielded 4,992 5' Sanger reads plus 673,811 GS FLX Titanium 454 sequences. After clustering S. pratensis yielded 12,000 unique transcripts, or unigenes, and C. orbicularis yielded 19,000. Both transcriptomes were very plant-like, i.e. most of the transcripts were more similar to streptophytes (land plants + charophyte green algae) than to other green algae in the sister group chlorophytes. BLAST results of several land plant genes hypothesized to be important in early land plant evolution resulted in high quality hits in both transcriptomes revealing putative orthologs ripe for follow-up studies.ConclusionsTwo main conclusions were drawn from this study. One illustrates the utility of next generation sequencing for transcriptome studies: larger scale data collection at a lower cost enabled us to cover a considerable portion of the transcriptome for both species. And, two, that the charophyte green algal transcriptoms are remarkably plant-like, which gives them the unique capacity to be major players for future evolutionary genomic studies addressing origin of land plant questions.

PHYLOGENY OF THE CONJUGATING GREEN ALGAE BASED ON CHLOROPLAST AND MITOCHONDRIAL NUCLEOTIDE SEQUENCE DATA(1).

The conjugating green algae represent a lineage of charophyte green algae known for their structural diversity and unusual mode of sexual reproduction, conjugation. These algae are ubiquitous in freshwater environments, where they are often important primary producers, but few studies have investigated evolutionary relationships in a molecular systematic context. A 109-taxon data set consisting of three gene fragments (two from the chloroplast and one from the mitochondrial genome) was used to estimate the phylogeny of the genera of the conjugating green algae. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) were used to estimate relationships from the 4,047 alignable nucleotides. This study confirmed the polyphyly of the Zygnemataceae and Mesotaeniaceae with respect to one another. The Peniaceae were determined to be paraphyletic, and two genera traditionally classified among the Zygnematales appear to belong to the lineage that gave rise to the Desmidiales. Six genera, Euastrum, Cosmarium, Cylindrocystis, Mesotaenium, Spondylosium, and Staurodesmus, were polyphyletic in this analysis. These findings have important implications for the evolution of structural characteristics in the group and will require some taxonomic changes. More work will be required to delineate lineages of Zygnematales in particular and to identify structural synapomorphies for some of the newly identified clades.

Nuclear DNA content estimates in green algal lineages: chlorophyta and streptophyta.

Consensus higher-level molecular phylogenies present a compelling case that an ancient divergence separates eukaryotic green algae into two major monophyletic lineages, Chlorophyta and Streptophyta, and a residuum of green algae, which have been referred to prasinophytes or micromonadophytes. Nuclear DNA content estimates have been published for less than 1% of the described green algal members of Chlorophyta, which includes multicellular green marine algae and freshwater flagellates (e.g. Chlamydomonas and Volvox). The present investigation summarizes the state of our knowledge and adds substantially to our database of C-values, especially for the streptophyte charophycean lineage which is the sister group of the land plants. A recent list of 2C nuclear DNA contents for isolates and species of green algae is expanded by 72 to 157. The DNA-localizing fluorochrome DAPI (4',6-diamidino-2-phenylindole) and red blood cell (chicken erythrocytes) standard were used to estimate 2C values with static microspectrophotometry. In Chlorophyta, including Chlorophyceae, Prasinophyceae, Trebouxiophyceae and Ulvophyceae, 2C DNA estimates range from 0.01 to 5.8 pg. Nuclear DNA content variation trends are noted and discussed for specific problematic taxon pairs, including Ulotrichales-Ulvales, and Cladophorales-Siphonocladales. For Streptophyta, 2C nuclear DNA contents range from 0.2 to 6.4 pg, excluding the highly polyploid Charales and Desmidiales, which have genome sizes of up to 14.8 and 46.8 pg, respectively. Nuclear DNA content data for Streptophyta superimposed on a contemporary molecular phylogeny indicate that early diverging lineages, including some members of Chlorokybales, Coleochaetales and Klebsormidiales, have genomes as small as 0.1-0.5 pg. It is proposed that the streptophyte ancestral nuclear genome common to both the charophyte and the embryophyte lineages can be characterized as 1C = 0.2 pg and 1n = 6. These data will help pre-screen candidate species for the on-going construction of bacterial artificial chromosome nuclear genome libraries for land plant ancestors. Data for the prasinophyte Mesostigma are of particular interest as this alga reportedly most closely resembles the 'ancestral green flagellate'. Both mechanistic and ecological processes are discussed that could have produced the observed C-value increase of >100-fold in the charophyte green algae whereas the ancestral genome was conserved in the embryophytes.

The complete mitochondrial DNA sequence of Mesostigma viride identifies this green alga as the earliest green plant divergence and predicts a highly compact mitochondrial genome in the ancestor of all green plants.

To gain insights into the nature of the mitochondrial genome in the common ancestor of all green plants, we have completely sequenced the mitochondrial DNA (mtDNA) of Mesostigma viride. This green alga belongs to a morphologically heterogeneous class (Prasinophyceae) that includes descendants of the earliest diverging green plants. Recent phylogenetic analyses of ribosomal RNAs (rRNAs) and concatenated proteins encoded by the chloroplast genome identified Mesostigma as a basal branch relative to the Streptophyta and the Chlorophyta, the two phyla that were previously thought to contain all extant green plants. The circular mitochondrial genome of Mesostigma resembles the mtDNAs of green algae occupying a basal position within the Chlorophyta in displaying a small size (42,424 bp) and a high gene density (86.6% coding sequences). It contains 65 genes that are conserved in other mtDNAs. Although none of these genes represents a novel coding sequence among green plant mtDNAs, four of them (rps1, sdh3, sdh4, and trnL[caa]) have not been reported previously in chlorophyte mtDNAs, and two others (rpl14 and trnI[gau]) have not been identified in the streptophyte mtDNAs examined so far (land-plant mtDNAs). Phylogenetic analyses of 19 concatenated mtDNA-encoded proteins favor the hypothesis that Mesostigma represents the earliest branch of green plant evolution. Four group I introns (two in rnl and two in cox1) and three group II introns (two in nad3 and one in cox2), two of which are trans-spliced at the RNA level, reside in Mesostigma mtDNA. The insertion sites of the three group II introns are unique to this mtDNA, suggesting that trans-splicing arose independently in the Mesostigma lineage and in the Streptophyta. The few structural features that can be regarded as ancestral in Mesostigma mtDNA predict that the common ancestor of all green plants had a compact mtDNA containing a minimum of 75 genes and perhaps two group I introns. Considering that the mitochondrial genome is much larger in size in land plants than in Mesostigma, we infer that mtDNA size began to increase dramatically in the Streptophyta either during the evolution of charophyte green algae or during the transition from charophytes to land plants.