The Charophycean green algae as model systems to study plant cell walls and other evolutionary adaptations that gave rise to land plants

The Charophycean green algae (CGA) occupy a key phylogenetic position as the evolutionary grade that includes the sister group of the land plants (embryophytes), and so provide potentially valuable experimental systems to study the development and evolution of traits that were necessary for terrestrial colonization. The nature and molecular bases of such traits are still being determined, but one critical adaptation is thought to have been the evolution of a complex cell wall. Very little is known about the identity, origins and diversity of the biosynthetic machinery producing the major suites of structural polymers (i. e., cell wall polysaccharides and associated molecules) that must have been in place for land colonization. However, it has been suggested that the success of the earliest land plants was partly based on the frequency of gene duplication, and possibly whole genome duplications, during times of radical habitat changes. Orders of the CGA span early diverging taxa retaining more ancestral characters, through complex multicellular organisms with morphological characteristics resembling those of land plants. Examination of gene diversity and evolution within the CGA could help reveal when and how the molecular pathways required for synthesis of key structural polymers in land plants arose.     

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.Key words: Chlorophyta, Streptophyta, Embryophyta, Charales, Coleochaetales, Zygnematales, viridiplant phylogeny, land plants, genome evolution, freshwater adaptation, sporophyte origin, diversification, extinction     

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.     

The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara corallina

Background and Aims

The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.

Methods

Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development.

Key Results

Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells.

Conclusions

Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.     

Chloroplast gene arrangement variation within a closely related group of green algae (Trebouxiophyceae, Chlorophyta)

The 22 published chloroplast genomes of green algae, representing sparse taxonomic sampling of diverse lineages that span over one billion years of evolution, each possess a unique gene arrangement. In contrast, many of the >190 published embryophyte (land plant) chloroplast genomes have relatively conserved architectures. To determine the phylogenetic depth at which chloroplast gene rearrangements occur in green algae, a 1.5-4 kb segment of the chloroplast genome was compared across nine species in three closely related genera of Trebouxiophyceae (Chlorophyta). In total, four distinct gene arrangements were obtained for the three genera Elliptochloris, Hemichloris, and Coccomyxa. In Elliptochloris, three distinct chloroplast gene arrangements were detected, one of which is shared with members of its sister genus Hemichloris. Both species of Coccomyxa examined share the fourth arrangement of this genome region, one characterized by very long spacers. Next, the order of genes found in this segment of the chloroplast genome was compared across green algae and land plants. As taxonomic ranks are not equivalent among different groups of organisms, the maximum molecular divergence among taxa sharing a common gene arrangement in this genome segment was compared. Well-supported clades possessing a single gene order had similar phylogenetic depth in green algae and embryophytes. When the dominant gene order of this chloroplast segment in embryophytes was assumed to be ancestral for land plants, the maximum molecular divergence was found to be over two times greater in embryophytes than in trebouxiophyte green algae. This study greatly expands information about chloroplast genome variation in green algae, is the first to demonstrate such variation among congeneric green algae, and further illustrates the fluidity of green algal chloroplast genome architecture in comparison to that of many embryophytes.     

Green algae to land plants: An evolutionary transition

Studies focused upon the evolutionary transition from ancestral green algae to the earliest land plants are important from a range of ecological, molecular and evolutionary perspectives. A substantial suite of ultrastructural, biochemical and molecular data supports the concept that land plants (embryophytes) are monophyletically derived from an ancestral charophycean alga. However, the details of phylogenetic branching patterns linking extant charophytes and seedless embryophytes are currently unclear. Moreover, the fossil record has so far been mute regarding the algae-land plant transition. Nevertheless, an accurate reflection of major evolutionary events in the history of the earliest land plants can be obtained by comparative paleontological-neontological studies, and comparative molecular, cellular and developmental investigations of extant charophytes and bryophytes. This review focuses upon research progress toward understanding three clade-specific adaptations that were important in the successful colonization of land by plants: the histogenetic apical meristem, the matrotrophic embryo, and decay-resistant cell wall polymers.     

Chlorogenic Acid Metabolism: The Evolution and Roles in Plant Response to Abiotic Stress

During the evolution, plants acquired the ability to synthesize different phenylpropanoid compounds like chlorogenic acid (CGA), which plays vital roles in resistance mechanisms to abiotic stresses. These environmental factors, including heavy metal, cold, heat, ultraviolet (UV) light, drought, and salinity affect the plant physiological processes, resulting in massive losses of agriculture production. As plants evolve from green algae to bryophytes, ferns, gymnosperms and angiosperms, phenylpropanoids are produced and accumulated in different tissues, giving the plant the capacity to counteract the harmful effects of the adverse environments. Studies have been performed on the metabolic evolution of rosmarinic acid, flavonoids and lignin, showing that the biosynthesis of phenylpropanoids begins in green algae until the emersion of genes found in angiosperms; however, the evolution of the CGA pathway has not yet been reviewed. We hypothesize that CGA could also be synthesized from algae to angiosperms. In the present review, the evolutionary analysis of CGA pathway and the function of this compound in plant tolerance to abiotic stresses are summarized. Bioinformatics analyzes were carried out on CGA-related genes across 37 plant species and revealed that the metabolic pathway starts in algae and gradually increases until it becomes complete in angiosperms. The key genes exhibited different expression patterns in stress and plant tissues. Interestingly, some genes accumulated rapidly during evolution and were more sensitive to environmental stresses, while others appeared only later in angiosperms. Further studies are needed to better understand the evolution of the CGA metabolic pathway in plants under environmentally stressed conditions.     

Occurrence and phylogenetic significance of cytokinesis-related callose in green algae, bryophytes, ferns and seed plants

 In order to investigate the occurrence of callose in dividing cells, we cultivated a selection of 30 organisms (the prokaryotic cyanobacterium Anabaena and eukaryotic green algae, bryophytes, ferns and seed plants) under defined conditions in the laboratory. Samples from these photoautotrophs, which are members of the evolutionary 'green lineage' leading from freshwater algae to land plants, were analysed by fluorescence microscopy. The β-1,3-glucan callose was identified by its staining properties with aniline blue and sirofluor. With the exception of the prokaryotic cyanobacterium, all of the eukaryotic organisms studied were capable of producing wound-induced callose. No callose was detected during cytokinesis of dividing cells of unicellular green algae (and Anabaena). However, in all of the multicellular green algae and land plants (embryophytes) investigated, callose was identified in newly made septae by an intense yellow fluorescence. The formation of wound callose was never detected in cells with callose in the newly formed septae. Additional experiments verified that no fixation-induced artefacts occurred. Our results show that callose is a regular component of developing septae in juvenile cells during cytokinesis in multicellular green algae and embryophytes. The implications of our results with respect to the evolutionary relationships between extant charophytes and land plants are discussed. Received: 15 September 2000 / Revision received: 23 October 2000 / Accepted: 23 October 2000     

OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF “SPECIAL WALLS” AT MEIOSPOROGENESIS IN COLEOCHAETE

Within germinating zygotes of Coleochaete pulvinata, meiospores are individually surrounded by chamber walls which are ultrastructurally and chemically different from vegetative cell walls of the same species. Meiospore chamber walls exhibit the staining reactions typical of callose. They thus resemble the “special walls” present during sporogenesis in embryophytes. Their presence suggests that the charophycean green algal ancestors of land plants may have possessed spore development preadaptations influential in the evolution of walled spores, an important plant adaptation to terrestrial life.     

Early evolution of life cycles in embryophytes:A focus on the fossil evidence of gametophyte/sporophyte size and morphological complexity

Embryophytes (land plants) are distinguished from their green algal ancestors by diplobiontic life cycles,that is,alternation of multicellular gametophytic and sporophytic generations.The bryophyte sporophyte is small and matrotrophic on the dominant gametophyte; extant vascular plants have an independent,dominant sporophyte and a reduced gametophyte.The elaboration of the diplobiontic life cycle in embryophytes has been thoroughly discussed within the context of the Antithetic and the Homologous Theories.The Antithetic Theory proposes a green algal ancestor with a gametophyte-dominant haplobiontic life cycle.The Homologous Theory suggests a green algal ancestor with alternation of isomorphic generations.The shifts that led from haplobiontic to diplobiontic life cycles and from gametophytic to sporophytic dominance are most probably related with terrestrial habitats.Cladistic studies strongly support the Antithetic Theory in repeatedly identifying charophycean green algae as the closest relatives of land plants.In recent years,exceptionally well-preserved axial gametophytes have been described from the Rhynie chert (Lower Devonian,410 Ma),and the complete life cycle of several Rhynie chert plants has been reconstructed.All show an alternation of more or less isomorphic generations,which is currently accepted as the plesiomorphic condition among all early polysporangiophytes,including basal tracheophytes.Here we review the existing evidence for early embryophyte gametophytes.We also discuss some recently discovered plants preserved as compression fossils and interpreted as gametophytes.All the fossil evidence supports the Antithetic Theory and indicates that the gametophytic generation/sporophytic generation size and complexity ratios show a gradual decrease along the land plant phylogenetic tree.     

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.     

Unearthing the molecular phylodiversity of desert soil green algae (Chlorophyta)

Deserts are not usually considered biodiversity hotspots, but desert microbiotic crust communities exhibit a rich diversity of both eukaryotic and prokaryotic life forms. Like many communities dominated by microscopic organisms, they defy characterization by traditional species-counting approaches to assessing biodiversity. Here we use exclusive molecular phylodiversity (E) to quantify the amount of evolutionary divergence unique to desert-dwelling green algae (Chlorophyta) in microbiotic crust communities. Given a phylogenetic tree with branch lengths expressed in units of expected substitutions per site, E is the total length of all tree segments representing exclusively desert lineages. Using MCMC to integrate over tree topologies and branch lengths provides 95% Bayesian credible intervals for phylodiversity measures. We found substantial exclusive molecular phylodiversity based on 18S rDNA data, showing that desert lineages are distantly related to their nearest aquatic relatives. Our results challenge conventional wisdom, which holds that there was a single origin of terrestrial green plants and that green algae are merely incidental visitors rather than indigenous components of desert communities. We identify examples of lineage diversification within deserts and at least 12 separate transitions from aquatic to terrestrial life apart from the most celebrated transition leading to the embryophyte land plants. [Bayesian phylogenetics; biodiversity; exclusive molecular phylodiversity; microbiotic crusts.].     

Early evolution of life cycles in embryophytes: A focus on the fossil evidence of gametophyte/sporophyte size and morphological complexity

Abstract Embryophytes (land plants) are distinguished from their green algal ancestors by diplobiontic life cycles, that is, alternation of multicellular gametophytic and sporophytic generations. The bryophyte sporophyte is small and matrotrophic on the dominant gametophyte; extant vascular plants have an independent, dominant sporophyte and a reduced gametophyte. The elaboration of the diplobiontic life cycle in embryophytes has been thoroughly discussed within the context of the Antithetic and the Homologous Theories. The Antithetic Theory proposes a green algal ancestor with a gametophyte‐dominant haplobiontic life cycle. The Homologous Theory suggests a green algal ancestor with alternation of isomorphic generations. The shifts that led from haplobiontic to diplobiontic life cycles and from gametophytic to sporophytic dominance are most probably related with terrestrial habitats. Cladistic studies strongly support the Antithetic Theory in repeatedly identifying charophycean green algae as the closest relatives of land plants. In recent years, exceptionally well‐preserved axial gametophytes have been described from the Rhynie chert (Lower Devonian, 410 Ma), and the complete life cycle of several Rhynie chert plants has been reconstructed. All show an alternation of more or less isomorphic generations, which is currently accepted as the plesiomorphic condition among all early polysporangiophytes, including basal tracheophytes. Here we review the existing evidence for early embryophyte gametophytes. We also discuss some recently discovered plants preserved as compression fossils and interpreted as gametophytes. All the fossil evidence supports the Antithetic Theory and indicates that the gametophytic generation/sporophytic generation size and complexity ratios show a gradual decrease along the land plant phylogenetic tree.     

Land plant biochemistry

Biochemical studies have complemented ultrastructural and, subsequently molecular genetic evidence consistent with the Charophyceae being the closest extant algal relatives of the embryophytes. Among the genes used in such molecular phylogenetic studies is that rbcL) for the large subunit of ribulose bisphosphate carboxylase-oxygenase (RUBISCO). The RUBISCO of the embryophytes is derived, via the Chlorophyta. from that of the cyanobacteria. This clade of the molecular phylogeny of RUBISCO shows a range of kinetic characteristics, especially of CO2 affinities and of CO2/O2 selectivities. The range of these kinetic values within the bryophytes is no greater than in the rest of the embryophytes; this has implications for the evolution of the embryophytes in the high atmospheric CO2 environment of the late Lower Palaeozoic. The differences in biochemistry between charophycean algae and embryophytes can to some extent be related functionally to the structure and physiology of embryophytes. Examples of components of embryophytes, which are qualitatively or quantitatively different from those of charophytes, are the water repellent/water resistant extracellular lipids, the rigid phenolic polymers functional in water-conducting elements and mechanical support in air, and in UV-B absorption, flavonoid phenolics involved in UV-B absorption and in interactions with other organisms, and the greater emphasis on low Mr organic acids. retained in the plant as free acids or salts, or secreted to the rhizosphere. The roles of these components are discussed in relation to the environmental conditions at the time of evolution of the terrestrial embryophytes. A significant point about embryophytes is the predominance of nitrogen-free extracellular structural material (a trait shared by most algae) and UV-B screening components, by contrast with analogous components in many other organisms. An important question, which has thus far been incompletely addressed, is the extent to which the absence from bryophytes of the biochemical pathways which produce components found only in tracheophytes is the result of evolutionary loss of these functions.     

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

BACKGROUND AND AIMS: 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. METHODS: 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. Key RESULTS: 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. CONCLUSIONS: 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 terrestrial biota prior to the origin of land plants (embryophytes): a review of the evidence

It is often assumed that life originated and diversified in the oceans prior to colonizing the land. However, environmental constraints in chemical evolution models point towards critical steps leading to the origin of life as having occurred in subaerial settings. The earliest fossil record does not include finds from terrestrial deposits, so much of our understanding about the presence of a terrestrial microbial cover prior to the Proterozoic is based on inference and geochemical proxies that indicate biospheric carbon cycling during the Archaean. Our assessment is that by 2.7 Ga, microbial ecosystems in terrestrial settings were driven by oxygen‐generating, photosynthetic cyanobacteria. Studies of modern organisms indicate that both the origin and primary diversification of the eukaryotes could have occurred in terrestrial settings, shortly after 2.0 Ga, but there is no direct fossil evidence of terrestrial eukaryotes until about 1.1 Ga. At this time, it appears that the diversity of life in non‐marine habitats exceeded that found in marine settings where sulphidic seas may have impaired eukaryotic physiology and retarded evolution. Geochemical proxies indicate the establishment of an extensive soil‐forming microbial cover by 850 Ma, and it is possible that a rise in atmospheric oxygen at this time was due to the evolutionary expansion of green algae into terrestrial habitats. Direct fossil evidence of the earliest terrestrial biotas in the Phanerozoic consists of problematical palynomorphs from the Cambro‐Ordovician of Laurentia. These indicate that the evolution of the first land plants (embryophytes) during the Middle Ordovician took place within a landscape that included aeroterrestrial algae which were actively adapting to selection in subaerial settings.     

Glycosyltransferase Family 43 Is Also Found in Early Eukaryotes and Has Three Subfamilies in Charophycean Green Algae

The glycosyltransferase family 43 (GT43) has been suggested to be involved in the synthesis of xylans in plant cell walls and proteoglycans in animals. Very recently GT43 family was also found in Charophycean green algae (CGA), the closest relatives of extant land plants. Here we present evidence that non-plant and non-animal early eukaryotes such as fungi, Haptophyceae, Choanoflagellida, Ichthyosporea and Haptophyceae also have GT43-like genes, which are phylogenetically close to animal GT43 genes. By mining RNA sequencing data (RNA-Seq) of selected plants, we showed that CGA have evolved three major groups of GT43 genes, one orthologous to IRX14 (IRREGULAR XYLEM14), one orthologous to IRX9/IRX9L and the third one ancestral to all land plant GT43 genes. We confirmed that land plant GT43 has two major clades A and B, while in angiosperms, clade A further evolved into three subclades and the expression and motif pattern of A3 (containing IRX9) are fairly different from the other two clades likely due to rapid evolution. Our in-depth sequence analysis contributed to our overall understanding of the early evolution of GT43 family and could serve as an example for the study of other plant cell wall-related enzyme families.