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 Transition From Algae to Embryophytes: Chloroplast Phylogenomic Evidence (II)

The transition of plant life from aquatic algae to land plants was one of the major events in the history of life. However, in hypothesizing the evolutionary path of the transition, limited shared phenotypic characters in aquatic algae and land plants (embryophytes) have been a major hinderance. Chloroplast genomes contain characters useful in tracing evolutionary histories. Embryophyte chloroplast genomes are distinguished from algal cpDNAs by the presence of over 20 group II introns and three ribosomal protein operons (rpl23, clpP and 3?rps12 operons). These phylogenomic features indicate a phylogenetic relationship of charophytes and embryophytes. In addition to these operons and introns, the evolution of rRNA and psbB operon evolution of streptophyte lineages will be incorporated with major biological phenotypic features to produce a phylogenetic tree. Basal embryophytes, the antithetic hypothesis, monophyly of embryophytes, and paraphyly of charophytes will be discussed. Strepotophytes are classified into three major groups (basal streptophytes, mid‐divergent streptophytes and late divergent charophytes‐embryophytes).     

The charophycean green algae provide insights into the early origins of plant cell walls

Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.     

Green algal phylogeny

Studies on the fine structure of green algal cells in the 1970s fundamentally revised theories on the evolution of green algae (Division Chlorophyta) and their relation to higher and drier green plants (i.e. embryophytes or land plants). Recent molecular phylogenetic work has largely confirmed some rather unorthodox proposals about which of the green algae represent the closest living relatives of higher plants. Resolution of the most ancient divergences on the green algal-land plant lineage remains elusive because of the rapidity of these evolutionary radiations and because branch topology varies with the taxa and molecular sequences sampled (as well as method of analysis). Molecular analyses within green algal groups have reinforced the value of ultrastructural characters and challenged the use of vegetative form as on overriding feature in classification.     

Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4',5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structure-function studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution insights into the architecture and mechanistic underpinnings of phenylpropanoid metabolizing enzymes contribute to our understanding of the emergence and on-going evolution of specialized phenylpropanoid products, and underscore the molecular basis of metabolic biodiversity at the chemical level. Finally, the detailed knowledge of the structure, function and evolution of these enzymes of specialized metabolism provide a set of experimental templates for the enzyme and metabolic engineering of production platforms for diverse novel compounds with desirable dietary and medicinal properties.     

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.     

Evolution and diversity of the 2–oxoglutarate‐dependent dioxygenase superfamily in plants

The 2–oxoglutarate‐dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation/hydroxylation reactions. Despite their biochemical significance in metabolism, a systematic analysis of plant 2OGDs remains to be accomplished. We present a phylogenetic classification of 479 2OGDs in six plant models, ranging from green algae to angiosperms. These were classified into three classes – DOXA, DOXB and DOXC – based on amino acid sequence similarity. The DOXA class includes plant homologs of Escherichia coli AlkB, which is a prototype of 2OGD involved in the oxidative demethylation of alkylated nucleic acids and histones. The DOXB class is conserved across all plant taxa and is involved in proline 4–hydroxylation in cell wall protein synthesis. The DOXC class is involved in specialized metabolism of various phytochemicals, including phytohormones and flavonoids. The vast majority of 2OGDs from land plants were classified into the DOXC class, but only seven from Chlamydomonas, suggesting that this class has diversified during land plant evolution. Phylogenetic analysis assigned DOXC‐class 2OGDs to 57 phylogenetic clades. 2OGD genes involved in gibberellin biosynthesis were conserved among vascular plants, and those involved in flavonoid and ethylene biosynthesis were shared among seed plants. Several angiosperm‐specific clades were found to be involved in various lineage‐specific specialized metabolisms, but 31 of the 57 DOXC‐class clades were only found in a single species. Therefore, the evolution and diversification of DOXC‐class 2OGDs is partly responsible for the diversity and complexity of specialized metabolites in land plants.     

THE PINGUIOPHYCEAE CLASSIS NOVA, CHROMOPHYTE ALGAE PRODUCING LARGE AMOUNTS OF OMEGA-3 FATTY ACIDS

The streptophytes comprise the Charophyceae sensu Mattox and Stewart (a morphologically diverse group of fresh-water green algae) and the embryophytes (land plants). Several charophycean groups are currently recognized. These include the Charales, Coleochaetales, Chlorokybales, Klebsormidiales and Zygnemophyceae (Desmidiales and Zygnematales). Recently, SSU rRNA gene sequence data allied Mesostigma viride (Prasinophyceae) with the Streptophyta. Complete chloroplast sequence data, however, placed Mesostigma sister to all green algae, not with the streptophytes. Several morphological, ultrastructural and biochemical features unite these lineages into a monophyletic group including embryophytes, but evolutionary relationships among the basal streptophytes remain ambiguous. To date, numerous studies using SSU rRNA gene sequences have yielded differing phylogenies with varying degrees of support dependent upon taxon sampling and choice of phylogenetic method. Like SSU data, chloroplast DNA sequence data have been used to examine relationships within the Charales, Coleochaetales, Zygnemophyceae and embryophytes. Representatives of all basal streptophyte lineages have not been examined using chloroplast data in a single analysis. Phylogenetic analyses were performed using DNA sequences of rbc L (the genes encoding the large subunit of rubisco) and atp B (the beta-subunit of ATPase) to examine relationships of basal streptophyte lineages. Preliminary analyses placed the branch leading to Mesostigma as the basal lineage in the Streptophyta with Chlorokybus , the sole representative of the Chlorokybales, branching next. Klebsormidiales and the enigmatic genus Entransia were sister taxa. Sister to these, the Charales, Coleochaetales, embryophytes and Zygnemophyceae formed a monophyletic group with Charales and Coleochaetales sister to each other and this clade sister to the embryophytes.     

Genomic evidences for the existence of a phenylpropanoid metabolic pathway in Aspergillus oryzae

Plants interact with their environment by producing a diverse array of secondary metabolites. A majority of these compounds are phenylpropanoids and flavonoids which are valued for their medicinal and agricultural properties. The phenylpropanoid biosynthesis pathway proceeds with the basic C6-C3 carbon skeleton of phenylalanine, and involves a wide range of enzymes viz., phenylalanine ammonia lyase, coumarate hydroxylase, coumarate ligase, chalcone synthase, chalcone reductase and chalcone isomerase. Recently, bacteria have also been shown to contain homodimeric polyketide synthases belonging to the plant chalcone synthase superfamily linking the capabilities of plants and bacteria in the biosynthesis of flavonoids. We report here the presence of genes encoding the core enzymes of the phenylpropanoid pathway in an industrially useful fungus, Aspergillus oryzae. Although the assignment of enzyme function must be confirmed by further biochemical evidences, this work has allowed us to anticipate the phenylpropanoid metabolism profile in a filamentous fungus for the first time and paves way for research on identifying novel fungal flavonoid-like metabolites.     

PHYLOGENY OF THE BASAL LINEAGES OF STREPTOPHYTA BASED ON RBCL AND ATPB GENE SEQUENCE DATA

The streptophytes comprise the Charophyceae sensu Mattox and Stewart (a morphologically diverse group of fresh‐water green algae) and the embryophytes (land plants). Several charophycean groups are currently recognized. These include the Charales, Coleochaetales, Chlorokybales, Klebsormidiales and Zygnemophyceae (Desmidiales and Zygnematales). Recently, SSU rRNA gene sequence data allied Mesostigma viride (Prasinophyceae) with the Streptophyta. Complete chloroplast sequence data, however, placed Mesostigma sister to all green algae, not with the streptophytes. Several morphological, ultrastructural and biochemical features unite these lineages into a monophyletic group including embryophytes, but evolutionary relationships among the basal streptophytes remain ambiguous. To date, numerous studies using SSU rRNA gene sequences have yielded differing phylogenies with varying degrees of support dependent upon taxon sampling and choice of phylogenetic method. Like SSU data, chloroplast DNA sequence data have been used to examine relationships within the Charales, Coleochaetales, Zygnemophyceae and embryophytes. Representatives of all basal streptophyte lineages have not been examined using chloroplast data in a single analysis. Phylogenetic analyses were performed using DNA sequences of rbcL (the genes encoding the large subunit of rubisco) and atpB (the beta‐subunit of ATPase) to examine relationships of basal streptophyte lineages. Preliminary analyses placed the branch leading to Mesostigma as the basal lineage in the Streptophyta with Chlorokybus, the sole representative of the Chlorokybales, branching next. Klebsormidiales and the enigmatic genus Entransia were sister taxa. Sister to these, the Charales, Coleochaetales, embryophytes and Zygnemophyceae formed a monophyletic group with Charales and Coleochaetales sister to each other and this clade sister to the embryophytes.     

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.     

Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H

Isoflavonoids are specialized plant metabolites, almost exclusive to legumes, and their biosynthesis forms a branch of the diverse phenylpropanoid pathway. Plant metabolism may be coordinated at many levels, including formation of protein complexes, or ‘metabolons’, which represent the molecular level of organization. Here, we have confirmed the existence of the long‐postulated isoflavonoid metabolon by identifying elements of the complex, their subcellular localizations and their interactions. Isoflavone synthase (IFS) and cinnamate 4–hydroxylase (C4H) have been shown to be tandem P450 enzymes that are anchored in the ER, interacting with soluble enzymes of the phenylpropanoid and isoflavonoid pathways (chalcone synthase, chalcone reductase and chalcone isomerase). The soluble enzymes of these pathways, whether localized to the cytoplasm or nucleus, are tethered to the ER through interaction with these P450s. The complex is also held together by interactions between the soluble elements. We provide evidence for IFS interaction with upstream and non‐consecutive enzymes. The existence of such a protein complex suggests a possible mechanism for flux of metabolites into the isoflavonoid pathway. Further, through interaction studies, we identified several candidates that are associated with GmIFS2, an isoform of IFS, in soybean hairy roots. This list provides additional candidates for various biosynthetic and structural elements that are involved in isoflavonoid production. Our interaction studies provide valuable information about isoform specificity among isoflavonoid enzymes, which may guide future engineering of the pathway in legumes or help overcome bottlenecks in heterologous expression.     

BUILDING A SPECIES PHYLOGENY FOR THE GENUS COLEOCHAETE (COLEOCHAETALES) USING THE GENES RBCL AND ATPB

The freshwater green alga Coleochaete Breb. (Coleochaetaceae; Coleochaetales) is a key streptophyte genus and is important to the understanding of the evolutionary origin of embryophytes (land plants). To date only a few species have been available from public culture collections. To facilitate research on this genus we have isolated 17 previously uncultured species of Coloechaete from material collected in the United States, Puerto Rico, and the Dominican Republic. Sequences for the genes rbcL and atpB were determined for these new isolates of Coleochaete (and for existing cultures) and combined with sequences from representative other streptophytes. Phylogenetic analyses indicate that Coleochaete, along with Chaetosphaeridium and Chara, are closely related to embryophytes and constitute a ‘higher streptophyte’ clade. At least four well‐supported lineages exist within Coleochaete. Characteristic growth forms have been identified for these four lineages, with important characters including aspects of thallus establishment, thallus habit, zygote development and hair sheath position. These data provide an improved understanding of species diversity and character evolution in the genus Coleochaete, and facilitate examination of hypotheses concerning character evolution in the streptophytes.     

Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis

The end products of the phenylpropanoid pathway play important roles in plant structure and development, as well as in plant defense mechanisms against biotic and abiotic stresses. From a human perspective, phenylpropanoid pathway-derived metabolites influence both human health and the potential utility of plants in agricultural contexts. The last known enzyme of the phenylpropanoid pathway that has not been characterized is p-coumarate 3-hydroxylase (C3H). By screening for plants that fail to accumulate soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. We have now shown that the ref8 mutant is defective in the gene encoding C3H. Phenotypic characterization of the ref8 mutant has revealed that the lack of C3H activity in the mutant leads to diverse changes in phenylpropanoid metabolism. The ref8 mutant accumulates p-coumarate esters in place of the sinapoylmalate found in wild-type plants. The mutant also deposits a lignin formed primarily from p-coumaryl alcohol, a monomer that is at best a minor component in the lignin of other plants. Finally, the mutant displays developmental defects and is subject to fungal attack, suggesting that phenylpropanoid pathway products downstream of REF8 may be required for normal plant development and disease resistance.