Metagenomic Analysis of Antarctic Biocrusts Unveils a Rich Range of Cold-Shock Proteins.

Microorganisms inhabiting Antarctic biocrusts develop several strategies to survive extreme environmental conditions such as severe cold and drought. However, the knowledge about adaptations of biocrusts microorganisms are limited. Here, we applied metagenomic sequencing to study biocrusts from east Antarctica. Biocrusts were dominated by cyanobacteria, actinobacteria and proteobacteria. Furthermore, the results provided insights into the presence and abundance of cold shock proteins (Csp), cold shock domain A proteins (CsdA), and antifreeze proteins (AFP) in these extreme environments. The metagenomic analysis revealed a high number of CsdA across the samples. The majority of the Csp recorded in the studied biocrusts were Csp A, C, and E. In addition, CsdA, Csp, and AFP primarily originated from proteobacteria and actinobacteria.

Metagenomics Provides a Deeper Assessment of the Diversity of Bacterial Communities in Polar Soils Than Metabarcoding.

The diversity of soil bacteria was analyzed via metabarcoding and metagenomic approaches using DNA samples isolated from the biocrusts of 12 different Arctic and Antarctic sites. For the metabarcoding approach, the V3-4 region of the 16S rRNA was targeted. Our results showed that nearly all operational taxonomic units (OTUs = taxa) found in metabarcoding analyses were recovered in metagenomic analyses. In contrast, metagenomics identified a large number of additional OTUs absent in metabarcoding analyses. In addition, we found huge differences in the abundance of OTUs between the two methods. The reasons for these differences seem to be (1) the higher sequencing depth in metagenomics studies, which allows the detection of low-abundance community members in metagenomics, and (2) bias of primer pairs used to amplify the targeted sequence in metabarcoding, which can change the community composition dramatically even at the lower taxonomic levels. We strongly recommend using only metagenomic approaches when establishing the taxonomic profiles of whole biological communities.

Microbial community composition of terrestrial habitats in East Antarctica with a focus on microphototrophs.

The Antarctic terrestrial environment harbors a diverse community of microorganisms, which have adapted to the extreme conditions. The aim of this study was to describe the composition of microbial communities in a diverse range of terrestrial environments (various biocrusts and soils, sands from ephemeral wetlands, biofilms, endolithic and hypolithic communities) in East Antarctica using both molecular and morphological approaches. Amplicon sequencing of the 16S rRNA gene revealed the dominance of Chloroflexi, Cyanobacteria and Firmicutes, while sequencing of the 18S rRNA gene showed the prevalence of Alveolata, Chloroplastida, Metazoa, and Rhizaria. This study also provided a comprehensive assessment of the microphototrophic community revealing a diversity of cyanobacteria and eukaryotic microalgae in various Antarctic terrestrial samples. Filamentous cyanobacteria belonging to the orders Oscillatoriales and Pseudanabaenales dominated prokaryotic community, while members of Trebouxiophyceae were the most abundant representatives of eukaryotes. In addition, the co-occurrence analysis showed a prevalence of positive correlations with bacterial taxa frequently co-occurring together.

Temperature- and light stress adaptations in Zygnematophyceae: The challenges of a semi-terrestrial lifestyle.

Streptophyte green algae comprise the origin of land plants and therefore life on earth as we know it today. While terrestrialization opened new habitats, leaving the aquatic environment brought additional abiotic stresses. More-drastic temperature shifts and high light levels are major abiotic stresses in semi-terrestrial habitats, in addition to desiccation, which has been reviewed elsewhere. Zygnematophyceae, a species-rich class of streptophyte green algae, is considered a sister-group to embryophytes. They have developed a variety of avoidance and adaptation mechanisms to protect against temperature extremes and high radiation in the form of photosynthetically active and ultraviolet radiation (UV) radiation occurring on land. Recently, knowledge of transcriptomic and metabolomic changes as consequences of these stresses has become available. Land-plant stress-signaling pathways producing homologs of key enzymes have been described in Zygnematophyceae. An efficient adaptation strategy is their mat-like growth habit, which provides self-shading and protects lower layers from harmful radiation. Additionally, Zygnematophyceae possess phenolic compounds with UV-screening ability. Resting stages such as vegetative pre-akinetes tolerate freezing to a much higher extent than do young cells. Sexual reproduction occurs by conjugation without the formation of flagellated male gametes, which can be seen as an advantage in water-deficient habitats. The resulting zygospores possess a multilayer cell wall, contributing to their resistance to terrestrial conditions. Especially in the context of global change, understanding temperature and light tolerance is crucial.

Biocrusts from Iceland and Svalbard: Does microbial community composition differ substantially?

A wide range of microorganisms inhabit biocrusts of arctic and sub-arctic regions. These taxa live and thrive under extreme conditions and, moreover, play important roles in biogeochemical cycling. Nevertheless, their diversity and abundance remain ambiguous. Here, we studied microbial community composition in biocrusts from Svalbard and Iceland using amplicon sequencing and epifluorescence microscopy. Sequencing of 16S rRNA gene revealed the dominance of Chloroflexi in the biocrusts from Iceland and Longyearbyen, and Acidobacteria in the biocrusts from Ny-Ålesund and South Svalbard. Within the 18S rRNA gene sequencing dataset, Chloroplastida prevailed in all the samples with dominance of Trebouxiophyceae in the biocrusts from Ny-Ålesund and Embryophyta in the biocrusts from the other localities. Furthermore, cyanobacterial number of cells and biovolume exceeded the microalgal in the biocrusts. Community compositions in the studied sites were correlated to the measured chemical parameters such as conductivity, pH, soil organic matter and mineral nitrogen contents. In addition, co-occurrence analysis showed the dominance of positive potential interactions and, bacterial and eukaryotic taxa co-occurred more frequently together.

Desiccation tolerance in streptophyte algae and the algae to land plant transition: evolution of LEA and MIP protein families within the Viridiplantae.

The present review summarizes the effects of desiccation in streptophyte green algae, as numerous experimental studies have been performed over the past decade particularly in the early branching streptophyte Klebsormidium sp. and the late branching Zygnema circumcarinatum. The latter genus gives its name to the Zygenmatophyceae, the sister group to land plants. For both organisms, transcriptomic investigations of desiccation stress are available, and illustrate a high variability in the stress response depending on the conditions and the strains used. However, overall, the responses of both organisms to desiccation stress are very similar to that of land plants. We highlight the evolution of two highly regulated protein families, the late embryogenesis abundant (LEA) proteins and the major intrinsic protein (MIP) family. Chlorophytes and streptophytes encode LEA4 and LEA5, while LEA2 have so far only been found in streptophyte algae, indicating an evolutionary origin in this group. Within the MIP family, a high transcriptomic regulation of a tonoplast intrinsic protein (TIP) has been found for the first time outside the embryophytes in Z. circumcarinatum. The MIP family became more complex on the way to terrestrialization but simplified afterwards. These observations suggest a key role for water transport proteins in desiccation tolerance of streptophytes.

Metatranscriptomic and metabolite profiling reveals vertical heterogeneity within a Zygnema green algal mat from Svalbard (High Arctic).

Within streptophyte green algae Zygnematophyceae are the sister group to the land plants that inherited several traits conferring stress protection. Zygnema sp., a mat-forming alga thriving in extreme habitats, was collected from a field site in Svalbard, where the bottom layers are protected by the top layers. The two layers were investigated by a metatranscriptomic approach and GC-MS-based metabolite profiling. In the top layer, 6569 genes were significantly upregulated and 149 were downregulated. Upregulated genes coded for components of the photosynthetic apparatus, chlorophyll synthesis, early light-inducible proteins, cell wall and carbohydrate metabolism, including starch-degrading enzymes. An increase in maltose in the top layer and degraded starch grains at the ultrastructural levels corroborated these findings. Genes involved in amino acid, redox metabolism and DNA repair were upregulated. A total of 29 differentially accumulated metabolites (out of 173 identified ones) confirmed higher metabolic turnover in the top layer. For several of these metabolites, differential accumulation matched the transcriptional changes of enzymes involved in associated pathways. In summary, the findings support the hypothesis that in a Zygnema mat the top layer shields the bottom layers from abiotic stress factors such as excessive irradiation.

Cold Acclimation Improves the Desiccation Stress Resilience of Polar Strains of Klebsormidium (Streptophyta).

Biological soil crusts (BSCs) are complex communities of autotrophic, heterotrophic, and saprotrophic (micro)organisms. In the polar regions, these biocrust communities have essential ecological functions such as primary production, nitrogen fixation, and ecosystem engineering while coping with extreme environmental conditions (temperature, desiccation, and irradiation). The microalga Klebsormidium is commonly found in BSCs all across the globe. The ecophysiological resilience of various Klebsormidium species to desiccation and other stresses has been studied intensively. Here we present the results of transcriptomic analyses of two different Klebsormidium species, K. dissectum and K. flaccidum, isolated from Antarctic and Arctic BSCs. We performed desiccation stress experiments at two different temperatures mimicking fluctuations associated with global change. Cultures grown on agar plates were desiccated on membrane filters at 10% relative air humidity until the photosynthetic activity as reflected in the effective quantum yield of photosystem II [Y(II)] ceased. For both species, the response to dehydration was much faster at the higher temperature. At the transcriptome level both species responded more strongly to the desiccation stress at the higher temperature suggesting that adaptation to cold conditions enhanced the resilience of both algae to desiccation stress. Interestingly, the two different species responded differently to the applied desiccation stress with respect to the number as well as function of genes showing differential gene expression. The portion of differentially expressed genes shared between both taxa was surprisingly low indicating that both Klebsormidium species adapted independently to the harsh conditions of Antarctica and the Arctic, respectively. Overall, our results indicate that environmental acclimation has a great impact on gene expression and the response to desiccation stress in Klebsormidium.

Biodiversity of biological soil crusts from the Polar Regions revealed by metabarcoding.

Biological soil crusts (BSCs) are amalgamations of autotrophic, heterotrophic and saprotrophic organisms. In the Polar Regions, these unique communities occupy essential ecological functions such as primary production, nitrogen fixation and ecosystem engineering. Here, we present the first molecular survey of BSCs from the Arctic and Antarctica focused on both eukaryotes and prokaryotes as well as passive and active biodiversity. Considering sequence abundance, Bryophyta is among the most abundant taxa in all analyzed BSCs suggesting that they were in a late successional stage. In terms of algal and cyanobacterial biodiversity, the genera Chloromonas, Coccomyxa, Elliptochloris and Nostoc were identified in all samples regardless of origin confirming their ubiquitous distribution. For the first time, we found the chrysophyte Spumella to be common in polar BSCs as it was present in all analyzed samples. Co-occurrence analysis revealed the presence of sulfur metabolizing microbes indicating that BSCs also play an important role for the sulfur cycle. In general, phototrophs were most abundant within the BSCs but there was also a diverse community of heterotrophs and saprotrophs. Our results show that BSCs are unique microecosystems in polar environments with an unexpectedly high biodiversity.

New barcoded primers for efficient retrieval of cercozoan sequences in high-throughput environmental diversity surveys, with emphasis on worldwide biological soil crusts.

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost-effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new-generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%-100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.

Enhanced Desiccation Tolerance in Mature Cultures of the Streptophytic Green Alga Zygnema circumcarinatum Revealed by Transcriptomics.

Desiccation tolerance is commonly regarded as one of the key features for the colonization of terrestrial habitats by green algae and the evolution of land plants. Extensive studies, focused mostly on physiology, have been carried out assessing the desiccation tolerance and resilience of the streptophytic genera Klebsormidium and Zygnema. Here we present transcriptomic analyses of Zygnema circumcarinatum exposed to desiccation stress. Cultures of Z. circumcarinatum grown in liquid medium or on agar plates were desiccated at ∼86% relative air humidity until the effective quantum yield of PSII [Y(II)] ceased. In general, the response to dehydration was much more pronounced in Z. circumcarinatum cultured in liquid medium for 1 month compared with filaments grown on agar plates for 7 and 12 months. Culture on solid medium enables the alga to acclimate to dehydration much better and an increase in desiccation tolerance was clearly correlated to increased culture age. Moreover, gene expression analysis revealed that photosynthesis was strongly repressed upon desiccation treatment in the liquid culture while only minor effects were detected in filaments cultured on agar plates for 7 months. Otherwise, both samples showed induction of stress protection mechanisms such as reactive oxygen species scavenging (early light-induced proteins, glutathione metabolism) and DNA repair as well as the expression of chaperones and aquaporins. Additionally, Z. circumcarinatum cultured in liquid medium upregulated sucrose-synthesizing enzymes and strongly induced membrane modifications in response to desiccation stress. These results corroborate the previously described hardening and associated desiccation tolerance in Zygnema in response to seasonal fluctuations in water availability.

The Origin and Evolution of the Plant Cell Surface: Algal Integrin-Associated Proteins and a New Family of Integrin-Like Cytoskeleton-ECM Linker Proteins.

The extracellular matrix of scaly green flagellates consists of small organic scales consisting of polysaccharides and scale-associated proteins (SAPs). Molecular phylogenies have shown that these organisms represent the ancestral stock of flagellates from which all green plants (Viridiplantae) evolved. The molecular characterization of four different SAPs is presented. Three SAPs are type-2 membrane proteins with an arginine/alanine-rich short cytoplasmic tail and an extracellular domain that is most likely of bacterial origin. The fourth protein is a filamin-like protein. In addition, we report the presence of proteins similar to the integrin-associated proteins α-actinin (in transcriptomes of glaucophytes and some viridiplants), LIM-domain proteins, and integrin-associated kinase in transcriptomes of viridiplants, glaucophytes, and rhodophytes. We propose that the membrane proteins identified are the predicted linkers between scales and the cytoskeleton. These proteins are present in many green algae but are apparently absent from embryophytes. These proteins represent a new protein family we have termed gralins for green algal integrins. Gralins are absent from embryophytes. A model for the evolution of the cell surface proteins in Plantae is discussed.

The impact of GSTM1/GSTT1 polymorphism for the risk of oral cancer.

OBJECTIVES: Since development of oral squamous cell cancer (OSCC) is triggered by various noxa, different variants of the antioxidant glutathione S-transferases (GSTs) can counteract toxic compounds (e.g., tobacco smoke). Because different polymorphisms of GST are known to have an increased sensitivity to carcinogenic agents, the aim of this study was to analyze whether GSTM1 or GSTT1 polymorphisms increase the risk for the development of OSCC. MATERIALS AND METHODS: GSTM1 and GSTT1 polymorphism was examined in healthy volunteers (n = 93) and in patients with OSCC (n = 100) by PCR after brush biopsy of oral mucosa. Odds ratio (OR) was calculated to evaluate the risk of oral cancer development. RESULTS: GSTM1 and GSTT1 deletion was found in 57% (53/93) and 18% (17/93), respectively, in healthy patients, while the OSCC group showed 57% (57/100) for GSTM1 deletion and 22% (22/100) with a deletion of GSTT1. Odds ratio for GSTM1 polymorphism was 1.00 and for GSTT1 1.26. Comparing smokers and nonsmokers with GSTM1 deletion polymorphism, OR was 4.35, while smokers without GSTM1 deletion showed an OR of 1.45. Adapting these data to the smoking habits of the general population in Germany, the OR was 9.25 for smokers with a GSTM1 deletion and OR 6.68 for smokers without a GSTM1 deletion. In smokers with GSTT1 deletion polymorphism, OR was 1.6 (adapted to the smoking habits of the general population: OR 6.16) and 3.16 (OR 8.56) in smokers without deletion in GSTT1 gene. CONCLUSIONS: Analysis of GST-M1 polymorphism in smokers could help to identify patients with a higher risk for the development of oral cancer. CLINICAL RELEVANCE: Early detection of OSCC due to a close meshed monitoring program for patients with GST-M1 polymorphism could help to improve the patient outcome. For polymorphism investigations, the oral brush biopsy is a sufficient method to gain DNA material.

Transcriptomics of desiccation tolerance in the streptophyte green alga Klebsormidium reveal a land plant-like defense reaction.

BACKGROUND: Water loss has significant effects on physiological performance and survival rates of algae. However, despite the prominent presence of aeroterrestrial algae in terrestrial habitats, hardly anything is known about the molecular events that allow aeroterrestrial algae to survive harsh environmental conditions. We analyzed the transcriptome and physiology of a strain of the alpine aeroterrestrial alga Klebsormidium crenulatum under control and strong desiccation-stress conditions. PRINCIPAL FINDINGS: For comparison we first established a reference transcriptome. The high-coverage reference transcriptome includes about 24,183 sequences (1.5 million reads, 636 million bases). The reference transcriptome encodes for all major pathways (energy, carbohydrates, lipids, amino acids, sugars), nearly all deduced pathways are complete or missing only a few transcripts. Upon strong desiccation, more than 7000 transcripts showed changes in their expression levels. Most of the highest up-regulated transcripts do not show similarity to known viridiplant proteins, suggesting the existence of some genus- or species-specific responses to desiccation. In addition, we observed the up-regulation of many transcripts involved in desiccation tolerance in plants (e.g. proteins similar to those that are abundant in late embryogenesis (LEA), or proteins involved in early response to desiccation ERD), and enzymes involved in the biosynthesis of the raffinose family of oligosaccharides (RFO) known to act as osmolytes). Major physiological shifts are the up-regulation of transcripts for photosynthesis, energy production, and reactive oxygen species (ROS) metabolism, which is supported by elevated cellular glutathione content as revealed by immunoelectron microscopy as well as an increase in total antiradical power. However, the effective quantum yield of Photosystem II and CO2 fixation decreased sharply under the applied desiccation stress. In contrast, transcripts for cell integrative functions such as cell division, DNA replication, cofactor biosynthesis, and amino acid biosynthesis were down-regulated. SIGNIFICANCE: This is the first study investigating the desiccation transcriptome of a streptophyte green alga. Our results indicate that the cellular response is similar to embryophytes, suggesting that embryophytes inherited a basic cellular desiccation tolerance from their streptophyte predecessors.

The contractile vacuole as a key regulator of cellular water flow in Chlamydomonas reinhardtii.

Most freshwater flagellates use contractile vacuoles (CVs) to expel excess water. We have used Chlamydomonas reinhardtii as a green model system to investigate CV function during adaptation to osmotic changes in culture medium. We show that the contractile vacuole in Chlamydomonas is regulated in two different ways. The size of the contractile vacuoles increases during cell growth, with the contraction interval strongly depending on the osmotic strength of the medium. In contrast, there are only small fluctuations in cytosolic osmolarity and plasma membrane permeability. Modeling of the CV membrane permeability indicates that only a small osmotic gradient is necessary for water flux into the CV, which most likely is facilitated by the aquaporin major intrinsic protein 1 (MIP1). We show that MIP1 is localized to the contractile vacuole, and that the expression rate and protein level of MIP1 exhibit only minor fluctuations under different osmotic conditions. In contrast, SEC6, a protein of the exocyst complex that is required for the water expulsion step, and a dynamin-like protein are upregulated under strong hypotonic conditions. The overexpression of a CreMIP1-GFP construct did not change the physiology of the CV. The functional implications of these results are discussed.

Snow ball earth and the split of Streptophyta and Chlorophyta.

About 700 million years ago (Mya), the ancestor of all green plants evolved into two major groups: the Chlorophyta (many green algae) and the Streptophyta (some green algae and land plants = embryophytes). Both groups are separated by several morphological, physiological, and molecular characteristics, including different photorespiration pathways. The Chloropyhta/Streptophyta split was probably very important for the colonization of the terrestrial habitat because embryophytes, the descendants of streptophyte algae, today completely dominate the macrophyte flora of the terrestrial habitats. By contrast, in aquatic ecosystems macrophytes from brown, red, and green algae compete with embryophytes. In this opinion article, I argue that the Chlorophyta/Streptophyta split is related to glaciation events (snow ball earth states) in the Neoproterozoic and provide an explanation for the different photorespiration pathways.

The SEC6 protein is required for contractile vacuole function in Chlamydomonas reinhardtii.

Contractile vacuoles (CVs) are essential for osmoregulation in many protists. To investigate the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory mutants. Four of the isolated mutant cell lines carried the same 33,641 base deletion, rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. The CV morphology was variable in mutant cells, and most cells had multiple small CVs. In addition, one or two enlarged CVs or no visible CVs at all, were observed by light microscopy. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants had long flagella. One of the affected genes is the only SEC6 homologue in Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex that is required for efficient exocytosis. Transformation of the Osmo75 mutant with a CreSEC6-GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains overexpressed CreSEC6 (as a GFP-tagged protein) and displayed a modified CV activity. CVs were larger, whereas the CV contraction interval remained unchanged, leading to increased water efflux rates. Electron microscopy analysis of Osmo75 cells showed that the mutant is able to form the close contact zones between the plasma membrane and the CV membrane observed during late diastole and systole. These results indicate that CreSEC6 is essential for CV function and required for homotypic vesicle fusion during diastole and water expulsion during systole. In addition, CreSEC6 is not only necessary for CV function, but possibly influences the CV cycle in an indirect manner and flagellar length in Chlamydomonas.

Origin of land plants: do conjugating green algae hold the key?

BACKGROUND: The terrestrial habitat was colonized by the ancestors of modern land plants about 500 to 470 million years ago. Today it is widely accepted that land plants (embryophytes) evolved from streptophyte algae, also referred to as charophycean algae. The streptophyte algae are a paraphyletic group of green algae, ranging from unicellular flagellates to morphologically complex forms such as the stoneworts (Charales). For a better understanding of the evolution of land plants, it is of prime importance to identify the streptophyte algae that are the sister-group to the embryophytes. The Charales, the Coleochaetales or more recently the Zygnematales have been considered to be the sister group of the embryophytes However, despite many years of phylogenetic studies, this question has not been resolved and remains controversial. RESULTS: Here, we use a large data set of nuclear-encoded genes (129 proteins) from 40 green plant taxa (Viridiplantae) including 21 embryophytes and six streptophyte algae, representing all major streptophyte algal lineages, to investigate the phylogenetic relationships of streptophyte algae and embryophytes. Our phylogenetic analyses indicate that either the Zygnematales or a clade consisting of the Zygnematales and the Coleochaetales are the sister group to embryophytes. CONCLUSIONS: Our analyses support the notion that the Charales are not the closest living relatives of embryophytes. Instead, the Zygnematales or a clade consisting of Zygnematales and Coleochaetales are most likely the sister group of embryophytes. Although this result is in agreement with a previously published phylogenetic study of chloroplast genomes, additional data are needed to confirm this conclusion. A Zygnematales/embryophyte sister group relationship has important implications for early land plant evolution. If substantiated, it should allow us to address important questions regarding the primary adaptations of viridiplants during the conquest of land. Clearly, the biology of the Zygnematales will receive renewed interest in the future.

The system of contractile vacuoles in the green alga Mesostigma viride (Streptophyta).

The contractile vacuole (CV) is an osmoregulatory organelle which is found in many protists. We have investigated the structure and function of the CV in the green alga Mesostigma viride by light (video) and serial section electron microscopy. Mesostigma is the only known flagellate streptophyte (charophycean green algae and land plants) and therefore of great importance for our understanding of the evolution of streptophytes. The entire CV system of Mesostigma has been reconstructed 3-dimensionally for three cells. Based on light microscopy cells contain an average of 8 CVs. The maximal diameter of a CV in Mesostigma was 1.5 microm and the expulsion interval 24.1s in 10 mosM medium. Video microscopy revealed the system of CVs to be very dynamic with individual CVs connecting temporarily or fusing completely with each other. Electron microscopy confirmed these observations and showed coated vesicles to be predominantly associated with large CVs. No expulsion pore was observed by electron microscopy. Instead we encountered close contact zones of approximately 150 nm diameter, which we propose to be the site of water expulsion. A model for the function of CVs in Mesostigma is presented.

Streptophyte algae and the origin of embryophytes.

BACKGROUND: Land plants (embryophytes) evolved from streptophyte green algae, a small group of freshwater algae ranging from scaly, unicellular flagellates (Mesostigma) to complex, filamentous thalli with branching, cell differentiation and apical growth (Charales). Streptophyte algae and embryophytes form the division Streptophyta, whereas the remaining green algae are classified as Chlorophyta. The Charales (stoneworts) are often considered to be sister to land plants, suggesting progressive evolution towards cellular complexity within streptophyte green algae. Many cellular (e.g. phragmoplast, plasmodesmata, hexameric cellulose synthase, structure of flagellated cells, oogamous sexual reproduction with zygote retention) and physiological characters (e.g. type of photorespiration, phytochrome system) originated within streptophyte algae. RECENT PROGRESS: Phylogenetic studies have demonstrated that Mesostigma (flagellate) and Chlorokybus (sarcinoid) form the earliest divergence within streptophytes, as sister to all other Streptophyta including embryophytes. The question whether Charales, Coleochaetales or Zygnematales are the sister to embryophytes is still (or, again) hotly debated. Projects to study genome evolution within streptophytes including protein families and polyadenylation signals have been initiated. In agreement with morphological and physiological features, many molecular traits believed to be specific for embryophytes have been shown to predate the Chlorophyta/Streptophyta split, or to have originated within streptophyte algae. Molecular phylogenies and the fossil record allow a detailed reconstruction of the early evolutionary events that led to the origin of true land plants, and shaped the current diversity and ecology of streptophyte green algae and their embryophyte descendants. CONCLUSIONS: The Streptophyta/Chlorophyta divergence correlates with a remarkably conservative preference for freshwater/marine habitats, and the early freshwater adaptation of streptophyte algae was a major advantage for the earliest land plants, even before the origin of the embryo and the sporophyte generation. The complete genomes of a few key streptophyte algae taxa will be required for a better understanding of the colonization of terrestrial habitats by streptophytes.