Photorespiration has been shown to be essential for all oxygenic phototrophs in the present-day oxygen-containing atmosphere. The strong similarity of the photorespiratory cycle in cyanobacteria and plants led to the hypothesis that oxygenic photosynthesis and photorespiration co-evolved in cyanobacteria, and then entered the eukaryotic algal lineages up to land plants via endosymbiosis. However, the evolutionary origin of the photorespiratory enzyme glycolate oxidase (GOX) is controversial, which challenges the common origin hypothesis. Here, we tested this hypothesis using phylogenetic and biochemical approaches with broad taxon sampling. Phylogenetic analysis supported the view that a cyanobacterial GOX-like protein of the 2-hydroxy-acid oxidase family most likely served as an ancestor for GOX in all eukaryotes. Furthermore, our results strongly indicate that GOX was recruited to the photorespiratory metabolism at the origin of Archaeplastida, because we verified that Glaucophyta, Rhodophyta, and Streptophyta all express GOX enzymes with preference for the substrate glycolate. Moreover, an “ancestral” protein synthetically derived from the node separating all prokaryotic from eukaryotic GOX-like proteins also preferred glycolate over l-lactate. These results support the notion that a cyanobacterial ancestral protein laid the foundation for the evolution of photorespiratory GOX enzymes in modern eukaryotic phototrophs.

Abstract Glaucophyta are members of the Archaeplastida, the founding group of photosynthetic eukaryotes that also includes red algae (Rhodophyta), green algae, and plants (Viridiplantae). Here we present a high-quality assembly, built using long-read sequences, of the ca. 100 Mb nuclear genome of the model glaucophyte Cyanophora paradoxa. We also conducted a quick-freeze deep-etch electron microscopy (QFDEEM) analysis of C. paradoxa cells to investigate glaucophyte morphology in comparison to other organisms. Using the genome data, we generated a resolved 115-taxon eukaryotic tree of life that includes a well-supported, monophyletic Archaeplastida. Analysis of muroplast peptidoglycan (PG) ultrastructure using QFDEEM shows that PG is most dense at the cleavage-furrow. Analysis of the chlamydial contribution to glaucophytes and other Archaeplastida shows that these foreign sequences likely played a key role in anaerobic glycolysis in primordial algae to alleviate ATP starvation under night-time hypoxia. The robust genome assembly of C. paradoxa significantly advances knowledge about this model species and provides a reference for exploring the panoply of traits associated with the anciently diverged glaucophyte lineage.

Polycomb Group (PcG) proteins mediate chromatin repression in plants and animals by catalyzing H3K27 methylation and H2AK118/119 mono-ubiquitination through the activity of the Polycomb repressive complex 2 (PRC2) and PRC1, respectively. PcG proteins were extensively studied in higher plants, but their function and target genes in unicellular branches of the green lineage remain largely unknown. To shed light on PcG function and modus operandi in a broad evolutionary context, we demonstrate phylogenetic relationship of core PRC1 and PRC2 proteins and H3K27me3 biochemical presence in several unicellular algae of different phylogenetic subclades. We focus then on one of the species, the model red alga Cyanidioschizon merolae, and show that H3K27me3 occupies both, genes and repetitive elements, and mediates the strength of repression depending on the differential occupancy over gene bodies. Furthermore, we report that H3K27me3 in C. merolae is enriched in telomeric and subtelomeric regions of the chromosomes and has unique preferential binding toward intein-containing genes involved in protein splicing. Thus, our study gives important insight for Polycomb-mediated repression in lower eukaryotes, uncovering a previously unknown link between H3K27me3 targets and protein splicing.

Cofactors such as NAD, AMP, and Coenzyme A (CoA) are essential for a diverse set of reactions and pathways in the cell. Specific carrier proteins are required to distribute these cofactors to different cell compartments, including peroxisomes. We previously identified a peroxisomal transport protein in Arabidopsis (Arabidopsis thaliana) called the peroxisomal NAD carrier (PXN). When assayed in vitro, this carrier exhibits versatile transport functions, e.g. catalyzing the import of NAD or CoA, the exchange of NAD/NADH, and the export of CoA. These observations raise the question about the physiological function of PXN in plants. Here, we used Saccharomyces cerevisiae to address this question. First, we confirmed that PXN, when expressed in yeast, is active and targeted to yeast peroxisomes. Secondl, detailed uptake analyses revealed that the CoA transport function of PXN can be excluded under physiological conditions due to its low affinity for this substrate. Third, we expressed PXN in diverse mutant yeast strains and investigated the suppression of the mutant phenotypes. These studies provided strong evidences that PXN was not able to function as a CoA transporter or a redox shuttle by mediating a NAD/NADH exchange, but instead catalyzed the import of NAD into peroxisomes against AMP in intact yeast cells.

The origin of oxygenic photosynthesis in the Archaeplastida common ancestor was foundational for the evolution of multicellular life. It is very likely that the primary endosymbiosis that explains plastid origin relied initially on the establishment of a metabolic connection between the host cell and captured cyanobacterium. We posit that these connections were derived primarily from existing host-derived components. To test this idea, we used phylogenomic and network analysis to infer the phylogenetic origin and evolutionary history of 37 validated plastid innermost membrane (permeome) metabolite transporters from the model plant Arabidopsis thaliana. Our results show that 57% of these transporter genes are of eukaryotic origin and that the captured cyanobacterium made a relatively minor (albeit important) contribution to the process. We also tested the hypothesis that the bacterium-derived hexose-phosphate transporter UhpC might have been the primordial sugar transporter in the Archaeplastida ancestor. Bioinformatic and protein localization studies demonstrate that this protein in the extremophilic red algae Galdieria sulphuraria and Cyanidioschyzon merolae are plastid targeted. Given this protein is also localized in plastids in the glaucophyte alga Cyanophora paradoxa, we suggest it played a crucial role in early plastid endosymbiosis by connecting the endosymbiont and host carbon storage networks. In summary, our work significantly advances understanding of plastid integration and favors a host-centric view of endosymbiosis. Under this view, nuclear genes of either eukaryotic or bacterial (noncyanobacterial) origin provided key elements of the toolkit needed for establishing metabolic connections in the primordial Archaeplastida lineage.

Plastids fulfill a variety of different functions (e.g., photosynthesis and amino acid biosynthesis) that rely on proteins of cyanobacterial (i.e., endosymbiont), noncyanobacterial, and 'host' (eukaryotic) origins. Analysis of plastid proteome data from glaucophytes and green algae allows robust inference of protein origins and organelle protein sharing across the >1 billion years of Archaeplastida evolution. Here, we show that more than one-third of genes encoding plastid proteins lack detectable homologs in Cyanobacteria, underlining the taxonomically broad contributions to plastid functions. Chlamydiae and Proteobacteria are the most significant other bacterial sources of plastid proteins. Mapping of plastid proteins to metabolic pathways shows a core set of anciently derived proteins in Archaeplastida, with many others being lineage specific and derived from independent horizontal gene transfer (HGT) events.

The endosymbiont hypothesis proposes that photosynthate from the cyanobiont was exported to the cytosol of the eukaryote host and polymerized from ADP-glucose into glycogen. Chlamydia-like pathogens are the second major source of foreign genes in Archaeplastida, suggesting that these obligate intracellular pathogens had a significant role during the establishment of endosymbiosis, likely through facilitating the metabolic integration between the endosymbiont and the eukaryotic host. In this opinion article, we propose that a hexose phosphate transporter of chlamydial origin was the first transporter responsible for exporting photosynthate out of the cyanobiont. This connection pre-dates the recruitment of the host-derived carbon translocators on the plastid inner membranes of green and red algae, land plants, and photosynthetic organisms of higher order endosymbiotic origin.

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.

The resurrection plant Craterostigma plantagineum is able to withstand desiccation of its vegetative tissues and is found in areas with variable water availability. The closely related species Lindernia brevidens and Lindernia subracemosa are both endemic to montane rainforests of coastal Africa, but remarkably L. brevidens is tolerant to desiccation. We studied the regulation of the desiccation-related LEA-like 11-24 gene at multiple levels in closely related species in order to investigate the conservation of mechanisms involved in desiccation tolerance. The dehydration-responsive transcription of the LEA-like 11-24 gene is differentially regulated in these plants. Comparison of the LEA-like 11-24 core promoter regions revealed that promoters have different activities, but some functional cis-acting elements are conserved between species. Upon dehydration, LEA-like 11-24 proteins are phosphorylated at different levels and phosphorylation sites are not conserved among the three LEA-like 11-24 proteins. Differences in the regulation of the LEA-like 11-24 gene in the studied plant species appear to be the result of mutations that occurred during evolution. We postulate that L. brevidens will eventually lose the ability to survive vegetative desiccation, given that this trait appears not to be essential for survival.

The engulfment of a photoautotrophic cyanobacterium by a primitive mitochondria-bearing eukaryote traces back to more than 1.2 billion years ago. This single endosymbiotic event not only provided the early petroalgae with the metabolic capacity to perform oxygenic photosynthesis, but also introduced a plethora of other metabolic routes ranging from fatty acids and amino acids biosynthesis, nitrogen and sulfur assimilation to secondary compounds synthesis. This implicated the integration and coordination of the newly acquired metabolic entity with the host metabolism. The interface between the host cytosol and the plastidic stroma became of crucial importance in sorting precursors and products between the plastid and other cellular compartments. The plastid envelope membranes fulfill different tasks: they perform important metabolic functions, as they are involved in the synthesis of carotenoids, chlorophylls, and galactolipids. In addition, since most genes of cyanobacterial origin have been transferred to the nucleus, plastidial proteins encoded by nuclear genes are post-translationally transported across the envelopes through the TIC–TOC import machinery. Most importantly, chloroplasts supply the photoautotrophic cell with photosynthates in form of reduced carbon. The innermost bilayer of the plastidic envelope represents the permeability barrier for the metabolites involved in the carbon cycle and is literally stuffed with transporter proteins facilitating their transfer. The intracellular metabolite transporters consist of polytopic proteins containing membrane spans usually in the number of four or more α-helices. Phylogenetic analyses revealed that connecting the plastid with the host metabolism was mainly a process driven by the host cell. In Arabidopsis, 58% of the metabolite transporters are of host origin, whereas only 12% are attributable to the cyanobacterial endosymbiont. This review focuses on the metabolite transporters of the inner envelope membrane of plastids, in particular the electrochemical potential-driven class of transporters. Recent advances in elucidating the plastidial complement of metabolite transporters are provided, with an update on phylogenetic relationship of selected proteins.

A particular adaptation to survival under limited water availability has been realized in the desiccation-tolerant resurrection plants, which tend to grow in a habitat with seasonal rainfall and long dry periods. One of the best-studied examples is Craterostigma plantagineum. Here we report an unexpected finding: Lindernia brevidens, a close relative of C. plantagineum, exhibits desiccation tolerance, even though it is endemic to the montane rainforests of Tanzania and Kenya, where it never experiences seasonal dry periods. L. brevidens has been found exclusively in two fragments of the ancient Eastern Arc Mountains, which were protected from the devastating Pleistocene droughts by the stable Indian Ocean temperature. Analysis of the microhabitat reveals that L. brevidens is found in the same habitat as hygrophilous plant species, which further indicates that the plant never dries out completely. The objective of this investigation was to address whether C. plantagineum and L. brevidens have desiccation-related pathways in common, or whether L. brevidens has acquired novel pathways. A third, closely related, desiccation-sensitive species, Lindernia subracemosa, has been included for comparison. Mechanisms that confer cellular protection during extreme water loss are well conserved between C. plantagineum and L. brevidens, including the interconversion of 2-octulose to sucrose within the two desiccation-tolerant species. Furthermore, transcriptional control regions of desiccation-related genes belonging to the late embryogenesis abundant (LEA) protein family are also highly conserved. We propose that L. brevidens is a neoendemic species that has retained desiccation tolerance through genome stability, despite tolerance being superfluous to environmental conditions.

Reversible protein phosphorylation/dephosphorylation is crucial for regulation of many cellular events, and increasing evidence indicates that this post-translational modification is also involved in the complex process of acquisition of desiccation tolerance. To analyze the phosphoproteome of the desiccation tolerant resurrection plant Craterostigma plantagineum, MOAC-enriched proteins from leaves at different stages of a de-/rehydration cycle were separated by 2-D PAGE and detected by phosphoprotein-specific staining. Using this strategy 20 putative phosphoproteins were identified by MALDI-TOF MS and MS/MS, which were not detected when total proteins were analyzed. The characterized desiccation-related phosphoproteins CDeT11-24 and CDeT6-19 were used as internal markers to validate the specificity of the analyses. For 16 of the identified proteins published evidence suggests that they are phosphoproteins. Comparative analysis of the 2-D gels showed that spot intensities of most identified putative phosphoproteins change during the de-/rehydration cycle. This suggests an involvement of these proteins in desiccation tolerance. Nearly all changes in the phosphoproteome of C. plantagineum, which are triggered by dehydration, are reversed within 4 days of rehydration, which is in agreement with physiological observations. Possible functions of selected proteins are discussed in the context of the de-/rehydration cycle.