Phylogeny of nuclear-encoded plastid-targeted proteins supports an early divergence of glaucophytes within Plantae

The phylogenetic position of the glaucophyte algae within the eukaryotic supergroup Plantae remains to be unambiguously established. Here, we assembled a multigene data set of conserved nuclear-encoded plastid-targeted proteins of cyanobacterial origin (i.e., through primary endosymbiotic gene transfer) from glaucophyte, red, and green (including land plants) algae to infer the branching order within this supergroup. We find strong support for the early divergence of glaucophytes within the Plantae, corroborating 2 important putatively ancestral characters shared by glaucophyte plastids and the cyanobacterial endosymbiont that gave rise to this organelle: the presence of a peptidoglycan deposition between the 2 organelle membranes and carboxysomes. Both these traits were apparently lost in the common ancestor of red and green algae after the divergence of glaucophytes.     

Pathway of cytosolic starch synthesis in the model glaucophyte Cyanophora paradoxa

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.     

Chlamydiae has contributed at least 55 genes to Plantae with predominantly plastid functions

Background

The photosynthetic organelle (plastid) originated via primary endosymbiosis in which a phagotrophic protist captured and harnessed a cyanobacterium. The plastid was inherited by the common ancestor of the red, green (including land plants), and glaucophyte algae (together, the Plantae). Despite the critical importance of primary plastid endosymbiosis, its ancient derivation has left behind very few “footprints” of early key events in organelle genesis.

Methodology/Principal Findings

To gain insights into this process, we conducted an in-depth phylogenomic analysis of genomic data (nuclear proteins) from 17 Plantae species to identify genes of a surprising provenance in these taxa, Chlamydiae bacteria. Previous studies show that Chlamydiae contributed many genes (at least 21 in one study) to Plantae that primarily have plastid functions and were postulated to have played a fundamental role in organelle evolution. Using our comprehensive approach, we identify at least 55 Chlamydiae-derived genes in algae and plants, of which 67% (37/55) are putatively plastid targeted and at least 3 have mitochondrial functions. The remainder of the proteins does not contain a bioinformatically predicted organelle import signal although one has an N-terminal extension in comparison to the Chlamydiae homolog. Our data suggest that environmental Chlamydiae were significant contributors to early Plantae genomes that extend beyond plastid metabolism. The chlamydial gene distribution and protein tree topologies provide evidence for both endosymbiotic gene transfer and a horizontal gene transfer ratchet driven by recurrent endoparasitism as explanations for gene origin.

Conclusions/Significance

Our findings paint a more complex picture of gene origin than can easily be explained by endosymbiotic gene transfer from an organelle-like point source. These data significantly extend the genomic impact of Chlamydiae on Plantae and show that about one-half (30/55) of the transferred genes are most closely related to sequences emanating from the genome of the only environmental isolate that is currently available. This strain, Candidatus Protochlamydia amoebophila UWE25 is an endosymbiont of Acanthamoeba and likely represents the type of endoparasite that contributed the genes to Plantae.     

Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan

Chloroplasts are believed to be descendants of ancestral cyanobacteria that had peptidoglycan layer between the outer and the inner membranes. Historically, the glaucophyte Cyanophora paradoxa and the rhizopod Paulinella chromatophora were believed to harbor symbiotic cyanobacteria having peptidoglycan, which were conventionally named “cyanelles”. In addition, the complete set of genes involved in the synthesis of peptidoglycan has been found in the moss Physcomitrella patens and some plants and algae. The presence of peptidoglycan-like structures was demonstrated by a new metabolic labeling technique in P. patens. However, many green algae and all known red algae lack peptidoglycan-related genes. That is the reason why we questioned the origin of peptidoglycan-synthesizing enzymes in the chloroplasts of the green algae and plants. We performed phylogenetic analysis of ten enzymes involved in the synthesis of peptidoglycan exploiting the Gclust homolog clusters and additional genomic data. As expected, all the identified genes encoded in the chromatophore genome of P. chromatophora were closely related to cyanobacterial homologs. In the green algae and plants, only two genes, murA and mraY, were found to be closely related to cyanobacterial homologs. The origins of all other genes were diverse. Unfortunately, the origins of C. paradoxa genes were not clearly determined because of incompleteness of published genomic data. We discuss on the probable evolutionary scenarios to explain the mostly non-cyanobacterial origins of the biosynthetic enzymes of chloroplast peptidoglycan: A plausible one includes extensive multiple horizontal gene transfers during the early evolution of Viridiplantae.     

Cyanobacterial contribution to algal nuclear genomes is primarily limited to plastid functions

A single cyanobacterial primary endosymbiosis that occurred approximately 1.5 billion years ago is believed to have given rise to the plastid in the common ancestor of the Plantae or Archaeplastida--the eukaryotic supergroup comprising red, green (including land plants), and glaucophyte algae. Critical to plastid establishment was the transfer of endosymbiont genes to the host nucleus (i.e., endosymbiotic gene transfer [EGT]). It has been postulated that plastid-derived EGT played a significant role in plant nuclear-genome evolution, with 18% (or 4,500) of all nuclear genes in Arabidopsis thaliana having a cyanobacterial origin with about one-half of these recruited for nonplastid functions. Here, we determine whether the level of cyanobacterial gene recruitment proposed for Arabidopsis is of the same magnitude in the algal sisters of plants by analyzing expressed-sequence tag (EST) data from the glaucophyte alga Cyanophora paradoxa. Bioinformatic analysis of 3,576 Cyanophora nuclear genes shows that 10.8% of these with significant database hits are of cyanobacterial origin and one-ninth of these have nonplastid functions. Our data indicate that unlike plants, early-diverging algal groups appear to retain a smaller number of endosymbiont genes in their nucleus, with only a minor proportion of these recruited for nonplastid functions.     

EEF2 analysis challenges the monophyly of Archaeplastida and Chromalveolata

Background

Classification of eukaryotes provides a fundamental phylogenetic framework for ecological, medical, and industrial research. In recent years eukaryotes have been classified into six major supergroups: Amoebozoa, Archaeplastida, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. According to this supergroup classification, Archaeplastida and Chromalveolata each arose from a single plastid-generating endosymbiotic event involving a cyanobacterium (Archaeplastida) or red alga (Chromalveolata). Although the plastids within members of the Archaeplastida and Chromalveolata share some features, no nucleocytoplasmic synapomorphies supporting these supergroups are currently known.

Methodology/Principal Findings

This study was designed to test the validity of the Archaeplastida and Chromalveolata through the analysis of nucleus-encoded eukaryotic translation elongation factor 2 (EEF2) and cytosolic heat-shock protein of 70 kDa (HSP70) sequences generated from the glaucophyte Cyanophora paradoxa, the cryptophytes Goniomonas truncata and Guillardia theta, the katablepharid Leucocryptos marina, the rhizarian Thaumatomonas sp. and the green alga Mesostigma viride. The HSP70 phylogeny was largely unresolved except for certain well-established groups. In contrast, EEF2 phylogeny recovered many well-established eukaryotic groups and, most interestingly, revealed a well-supported clade composed of cryptophytes, katablepharids, haptophytes, rhodophytes, and Viridiplantae (green algae and land plants). This clade is further supported by the presence of a two amino acid signature within EEF2, which appears to have arisen from amino acid replacement before the common origin of these eukaryotic groups.

Conclusions/Significance

Our EEF2 analysis strongly refutes the monophyly of the Archaeplastida and the Chromalveolata, adding to a growing body of evidence that limits the utility of these supergroups. In view of EEF2 phylogeny and other morphological evidence, we discuss the possibility of an alternative eukaryotic supergroup.     

Proteomic analysis of the Cyanophora paradoxa muroplast provides clues on early events in plastid endosymbiosis

Glaucophytes represent the first lineage of photosynthetic eukaryotes of primary endosymbiotic origin that diverged after plastid establishment. The muroplast of Cyanophora paradoxa represents a primitive plastid that resembles its cyanobacterial ancestor in pigment composition and the presence of a peptidoglycan wall. To attain insights into the evolutionary history of cyanobiont integration and plastid development, it would thus be highly desirable to obtain knowledge on the composition of the glaucophyte plastid proteome. Here, we provide the first proteomic analysis of the muroplast of C. paradoxa. Mass spectrometric analysis of the muroplast proteome identified 510 proteins with high confidence. The protein repertoire of the muroplast revealed novel paths for reduced carbon flow and export to the cytosol through a sugar phosphate transporter of chlamydial origin. We propose that C. paradoxa possesses a primordial plastid mirroring the situation in the early protoalga.     

A molecular timeline for the origin of photosynthetic eukaryotes

The appearance of photosynthetic eukaryotes (algae and plants) dramatically altered the Earth's ecosystem, making possible all vertebrate life on land, including humans. Dating algal origin is, however, frustrated by a meager fossil record. We generated a plastid multi-gene phylogeny with Bayesian inference and then used maximum likelihood molecular clock methods to estimate algal divergence times. The plastid tree was used as a surrogate for algal host evolution because of recent phylogenetic evidence supporting the vertical ancestry of the plastid in the red, green, and glaucophyte algae. Nodes in the plastid tree were constrained with six reliable fossil dates and a maximum age of 3,500 MYA based on the earliest known eubacterial fossil. Our analyses support an ancient (late Paleoproterozoic) origin of photosynthetic eukaryotes with the primary endosymbiosis that gave rise to the first alga having occurred after the split of the Plantae (i.e., red, green, and glaucophyte algae plus land plants) from the opisthokonts sometime before 1,558 MYA. The split of the red and green algae is calculated to have occurred about 1,500 MYA, and the putative single red algal secondary endosymbiosis that gave rise to the plastid in the cryptophyte, haptophyte, and stramenopile algae (chromists) occurred about 1,300 MYA. These dates, which are consistent with fossil evidence for putative marine algae (i.e., acritarchs) from the early Mesoproterozoic (1,500 MYA) and with a major eukaryotic diversification in the very late Mesoproterozoic and Neoproterozoic, provide a molecular timeline for understanding algal evolution.     

Host origin of plastid solute transporters in the first photosynthetic eukaryotes

Background  

It is generally accepted that a single primary endosymbiosis in the Plantae (red, green (including land plants), and glaucophyte algae) common ancestor gave rise to the ancestral photosynthetic organelle (plastid). Plastid establishment necessitated many steps, including the transfer and activation of endosymbiont genes that were relocated to the nuclear genome of the 'host' followed by import of the encoded proteins into the organelle. These innovations are, however, highly complex and could not have driven the initial formation of the endosymbiosis. We postulate that the re-targeting of existing host solute transporters to the plastid fore-runner was critical for the early success of the primary endosymbiosis, allowing the host to harvest endosymbiont primary production.     

Intron distribution in Plantae: 500 million years of stasis during land plant evolution

Little is known about the evolution of the intron-exon organization in the more primitive groups of land plants, and the intron distribution among Plantae (glauco-, rhodo-, chloro- and streptophytes) has not been investigated so far. The present study is focused on some key species such as the liverwort Marchantia polymorpha, representing the most ancient lineage of land plants, and the streptophycean green alga Mesostigma viride, branching prior to charophycean green algae and terrestrial plants. The intron distribution of six genes for sugar phosphate metabolism was analyzed including four different glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the sedoheptulose-1,7-bisphosphatase (SBP) and the glucose-6-phosphate isomerase (GPI). We established 15 new sequences including three cDNA and twelve genomic clones with up to 24 introns per gene, which were identified in the GPI of Marchantia. The intron patterns of all six genes are completely conserved among seed plants, lycopods, mosses and even liverworts. This intron stasis without any gain of novel introns seem to last for nearly 500 million years and may be characteristic for land plants in general. Some unique intron positions in Mesostigma document that a uniform distribution is no common trait of all streptophytes, but it may correlate with the transition to terrestrial habitats. However, the respective genes of chlorophycean green algae display largely different patterns, thus indicating at least one phase of massive intron rearrangement in the green lineage. We moreover included rhodophyte and glaucophyte reference sequences in our analyses and, even if the well documented monophyly of Plantae is not reflected by a uniform intron distribution, at least one GPI intron is strictly conserved for 1.5 billion years.     

Evolution of Tertiary Structure of Viral RNA Dependent Polymerases

Viral RNA dependent polymerases (vRdPs) are present in all RNA viruses; unfortunately, their sequence similarity is too low for phylogenetic studies. Nevertheless, vRdP protein structures are remarkably conserved. In this study, we used the structural similarity of vRdPs to reconstruct their evolutionary history. The major strength of this work is in unifying sequence and structural data into a single quantitative phylogenetic analysis, using powerful a Bayesian approach.The resulting phylogram of vRdPs demonstrates that RNA-dependent DNA polymerases (RdDPs) of viruses within Retroviridae family cluster in a clearly separated group of vRdPs, while RNA-dependent RNA polymerases (RdRPs) of dsRNA and +ssRNA viruses are mixed together. This evidence supports the hypothesis that RdRPs replicating +ssRNA viruses evolved multiple times from RdRPs replicating +dsRNA viruses, and vice versa. Moreover, our phylogram may be presented as a scheme for RNA virus evolution. The results are in concordance with the actual concept of RNA virus evolution. Finally, the methods used in our work provide a new direction for studying ancient virus evolution.     

The largest subunit of RNA polymerase II from the Glaucocystophyta: functional constraint and short-branch exclusion in deep eukaryotic phylogeny

Background  

Evolutionary analyses of the largest subunit of RNA polymerase II (RPB1) have yielded important and at times provocative results. One particularly troublesome outcome is the consistent inference of independent origins of red algae and green plants, at odds with the more widely accepted view of a monophyletic Plantae comprising all eukaryotes with primary plastids. If the hypothesis of a broader kingdom Plantae is correct, then RPB1 trees likely reflect a persistent phylogenetic artifact. To gain a better understanding of RNAP II evolution, and the presumed artifact relating to green plants and red algae, we isolated and analyzed RPB 1 from representatives of Glaucocystophyta, the third eukaryotic group with primary plastids.     

Small RNAs from cereal powdery mildew pathogens may target host plant genes

Small RNAs (sRNAs) play a key role in eukaryotic gene regulation, for example by gene silencing via RNA interference (RNAi). The biogenesis of sRNAs depends on proteins that are generally conserved in all eukaryotic lineages, yet some species that lack part or all the components of the mechanism exist. Here we explored the presence of the RNAi machinery and its expression as well as the occurrence of sRNA candidates and their putative endogenous as well as host targets in phytopathogenic powdery mildew fungi. We focused on the species Blumeria graminis, which occurs in various specialized forms (formae speciales) that each have a strictly limited host range. B. graminis f. sp. hordei and B. graminis f. sp. tritici, colonizing barley and wheat, respectively, have genomes that are characterized by extensive gene loss. Nonetheless, we find that the RNAi machinery appears to be largely complete and expressed during infection. sRNA sequencing data enabled the identification of putative sRNAs in both pathogens. While a considerable part of the sRNA candidates have predicted target sites in endogenous genes and transposable elements, a small proportion appears to have targets in planta, suggesting potential cross-kingdom RNA transfer between powdery mildew fungi and their respective plant hosts.     

Red and green algal monophyly and extensive gene sharing found in a rich repertoire of red algal genes

The Plantae comprising red, green (including land plants), and glaucophyte algae are postulated to have a single common ancestor that is the founding lineage of photosynthetic eukaryotes. However, recent multiprotein phylogenies provide little or no support for this hypothesis. This may reflect limited complete genome data available for red algae, currently only the highly reduced genome of Cyanidioschyzon merolae, a reticulate gene ancestry, or variable gene divergence rates that mislead phylogenetic inference. Here, using novel genome data from the mesophilic Porphyridium cruentum and Calliarthron tuberculosum, we analyze 60,000 novel red algal genes to test the monophyly of red + green (RG) algae and their extent of gene sharing with other lineages. Using a gene-by-gene approach, we find an emerging signal of RG monophyly (supported by ～50% of the examined protein phylogenies) that increases with the number of distinct phyla and terminal taxa in the analysis. A total of 1,808 phylogenies show evidence of gene sharing between Plantae and other lineages. We demonstrate that a rich mesophilic red algal gene repertoire is crucial for testing controversial issues in eukaryote evolution and for understanding the complex patterns of gene inheritance in protists.