Complex origins of chloroplast membranes with photosynthetic machineries: multiple transfers of genes from divergent organisms at different times or a single endosymbiotic event?

The paradigm “cyanobacterial origin of chloroplasts” is currently viewed as an established fact. However, we may have to re-consider the origin of chloroplast membranes, because membranes are not replicated by their own. It is the genes for lipid biosynthetic enzymes that are inherited. In the current understandings, these enzymes became encoded by the nuclear genome as a result of endosymbiotic gene transfer from the endosymbiont. However, we previously showed that many enzymes involved in the synthesis of chloroplast peptidoglycan and glycolipids did not originate from cyanobacteria. Here I present results of comprehensive phylogenetic analysis of chloroplast enzymes involved in fatty acid and lipid biosynthesis, as well as additional chloroplast components related to photosynthesis and gene expression. Four types of phylogenetic relationship between chloroplast enzymes (encoded by the chloroplast and nuclear genomes) and cyanobacterial counterparts were found: type 1, chloroplast enzymes diverged from inside of cyanobacterial clade; type 2, chloroplast and cyanobacterial enzymes are sister groups; type 3, chloroplast enzymes originated from homologs of bacteria other than cyanobacteria; type 4, chloroplast enzymes diverged from eukaryotic homologs. Estimation of evolutionary distances suggested that the acquisition times of chloroplast enzymes were diverse, indicating that multiple gene transfers accounted for the chloroplast enzymes analyzed. Based on the results, I try to relax the tight logic of the endosymbiotic origin of chloroplasts involving a single endosymbiotic event by proposing alternative hypotheses. The hypothesis of host-directed chloroplast formation proposes that glycolipid synthesis ability had been acquired by the eukaryotic host before the acquisition of chloroplast ribosomes. Chloroplast membrane system could have been provided by the host, whereas cyanobacteria contributed to the genes for the genetic and photosynthesis systems, at various times, either before or after the formation of chloroplast membranes. The origin(s) of chloroplasts seems to be more complicated than the single event of primary endosymbiosis.

     

Bacterial glycoglycerolipid synthases: processive and non-processive glycosyltransferases in mycoplasma

Glycoglycerolipids are abundant membrane components in the photosynthetic tissues of plants and in cyanobacteria, with highly conserved structures (galactolipids). In non-photosynthetic bacteria, glycoglycerolipids are also widespread but with higher structural diversity. They are synthesized by the action of glycosyltransferases (GT), which transfers a glycosyl unit from a sugar nucleotide donor to diacylglycerol to form monoglycosyldiacylglycerol followed by a second transfer to give diglycosyldiacylglycerol. Both transferase activities are catalysed by different GT enzymes in plants, and many bacteria; however, processive enzymes, in which a single GT transfers the first and second (and eventually more) glycosyl units are also found in some bacteria. In this review, we summarize the diversity of glycosyltransferases involved in glycolipid biosynthesis in bacteria, focussing on mycoplasma enzymes and comparing processive and non-processive glycolipid synthases. Since glycoglycerolipids are key structural components of the plasma membrane in mycoplasmas, the glycolipid synthases involved in their biosynthesis are proposed as targets for the design of new antibiotics against mycoplasma infections.     

Evolution of enzymes involved in the photorespiratory 2-phosphoglycolate cycle from cyanobacteria via algae toward plants

The photorespiratory pathway was shown to be essential for organisms performing oxygenic photosynthesis, cyanobacteria, algae, and plants, in the present day O(2)-containing atmosphere. The identification of a plant-like 2-phosphoglycolate cycle in cyanobacteria indicated that not only genes of oxygenic photosynthesis but also genes encoding photorespiratory enzymes were endosymbiotically conveyed from ancient cyanobacteria to eukaryotic oxygenic phototrophs. Here, we investigated the origin of the photorespiratory pathway in photosynthetic eukaryotes by phylogenetic analysis. We found that a mixture of photorespiratory enzymes of either cyanobacterial or α-proteobacterial origin is present in algae and higher plants. Three enzymes in eukaryotic phototrophs clustered closely with cyanobacterial homologs: glycolate oxidase, glycerate kinase, and hydroxypyruvate reductase. On the other hand, the mitochondrial enzymes of the photorespiratory cycle in algae and plants, glycine decarboxylase subunits and serine hydroxymethyltransferase, evolved from proteobacteria. Other than most genes for proteins of the photosynthetic machinery, nearly all enzymes involved in the 2-phosphogylcolate metabolism coexist in the genomes of cyanobacteria and heterotrophic bacteria.     

Function and evolution of grana

Chloroplasts are descended from cyanobacteria, and they retain many features of the cyanobacterial photosynthetic apparatus. However, land-plant chloroplasts have a strikingly different thylakoid membrane organization to that of cyanobacteria. Usually the two photosystems are laterally segregated; Photosystem II is concentrated in complex stacked-membrane structures known as grana. The function of grana has long been debated. Recent studies on membrane organization in chloroplasts, cyanobacteria and purple bacteria now offer a new perspective. I argue that grana allow the presence of a large light-harvesting antenna for Photosystem II, without excessively restricting electron transport. Other organisms solve this problem in different ways. Land plants evolved from macroalgae that were adapted to high light conditions; they evolved grana as a new solution to the problem of efficient photosynthesis in shade.     

Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression

Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles—chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems.     

Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis

The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged -323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution.     

Lipid synthesis and metabolism in the plastid envelope

Plastid envelope membranes play a major role in the biosynthesis of glycerolipids. In addition, plastids are characterized by the occurrence of plastid-specific membrane glycolipids (galactolipids, a sulfolipid). Plant lipid metabolism therefore has unique features, when compared to that of other eukaryotic organisms, such as animals and yeast. However, the glycerolipid biosynthetic pathway in chloroplasts is almost identical to that found in cyanobacteria, and reflects the prokaryotic origin of the chloroplast. Fatty acids generated in the plastid stroma are substrates for a whole set of enzymes involved in the synthesis of polar lipids of plastid membranes such as galactolipids, the sulfolipid, the phosphatidylglycerol. In addition, fatty acids are exported outside the plastid where they are used for extraplastidial polar lipid synthesis (phosphatidylcholine, phosphatidylethanolamine, etc.). Various desaturation steps leading to the formation of polyunsaturated fatty acids occur in various cell compartments, especially in chloroplasts, using fatty acids esterified to polar lipids as substrates. Furthermore, plant glycerolipids can be metabolized by a series of very active envelope enzymes, such as the galactolipid:galactolipid galactosyltransferase and the acyl-galactolipid forming enzyme. The physiological significance of these enzymes is however largely unknown. One of the most active pathways involved in lipid metabolism and present in envelope membranes is the oxylipin pathway: polyunsaturated fatty acids that are released from polar lipids under various conditions (injury, pathogen attack) are converted to oxylipin. Thus, the plastid envelope membranes are also involved in the formation of signalling molecules.     

Roles of the acidic lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol in photosynthesis: their specificity and evolution

Sulfoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG) are lipids with negative charges, distributed among membranes of chloroplasts of plants and their postulated progenitors, cyanobacteria, and also widely among membranes of anoxygenic photosynthetic bacteria. Thus, these acidic lipids are of great interest in terms of their roles in the function and evolution of the photosynthetic membranes. The physiological significance of these lipids in photosynthesis has been examined through characterization of mutants defective in their abilities to synthesize SQDG or PG, and through characterization of isolated thylakoid membranes or photosynthetic particles, the acidic lipid contents of which were manipulated in vitro, for example, on treatment with phospholipase to degrade PG. Responsibility of SQDG or PG has been clarified so far in terms of the structural and/or functional integrity of photosystems I and/or II in cyanobacterial, green algal, and higher plant species. Also implied were distinct levels of the responsibility in the different photosynthetic organisms. Extreme cases involved the indispensability of SQDG for photosynthesis and growth in two prokaryotic, photosynthetic organisms and the contribution of PG to construction of the photosystem-I trimer exclusively in cyanobacteria. Here, roles of these acidic lipids are discussed with a focus on their specificity and the evolution of photosynthetic membranes.Norihiro Sato is the recipient of the Botanical Society Award for Young Scientist, 2003.     

The evolutionary origin of red algae as deduced from the nuclear genes encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases from Chondrus crispus

Algae are a heterogeneous group of photosynthetic eukaryotes traditionally separated into three major subdivisions: rhodophytes, chlorophytes, and chromophytes. The evolutionary origin of rhodophytes or red algae and their links to other photosynthetic and nonphotosynthetic eukaryotes have been a matter of much controversy and speculation. Here we present the first cDNAs of nuclear protein genes from red algae: Those encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Chondrus crispus. A phylogenetic analysis including GAPDH gene sequences from a number of eukaryotic taxa, cyanobacteria, and purple bacteria suggests that chloroplasts and rhodoplasts together form a monophyletic group of cyanobacterial descent and that rhodophytes separated from chlorophytes at about the same time as animals and fungi. The composite GAPDH tree further demonstrates that chloroplast and cytosolic GAPDH genes are closely related to their homologs in cyanobacteria and purple bacteria, respectively, the presumptive ancestors of chloroplasts and mitochondria, thereby firmly establishing the endosymbiotic origin of these nuclear genes and their fixation in eukaryotic cells before the rhodophyte/chlorophyte separation. The present data are in conflict with phylogenetic inferences based on plastid-encoded rbcL sequences supporting a polyphyletic origin of rhodoplasts and chloroplasts. Comparison of rbcL to GAPDH phylogenies suggests that rbcL trees may be misleading because they are composed of branches representing ancient duplicated (paralogous) genes. Correspondence to: R. Cerff     

Do photosynthetic and respiratory electron transport chains share redox proteins?

In purple nonsulfur bacteria and cyanobacteria, there is close interaction between the photosynthetic and respiratory electron transport chains, which share identical redox proteins. Recent findings that the thylakoid membranes of eukaryotic chloroplasts may have respiratory functions suggest that the interaction of photosynthesis and respiration may be a common feature of all photosynthetic cells.     

Protein phosphorylation and control of excitation energy transfer in photosynthetic purple bacteria and cyanobacteria

The function of phosphorylation of light-harvesting polypeptides is well characterised in chloroplasts of green plants, but the prokaryotic cyanobacteria and purple photosynthetic bacteria have quite different light-harvesting polypeptides whose structure and function cannot be controlled in precisely the same way. Nevertheless, cyanobacteria show light-dependent phosphorylation of membrane polypeptides associated with photosystem II and with the light-harvesting phycobilisome, and purple bacteria show light-dependent phosphorylation of low molecular-weight chromatophore membrane polypeptides. In both cases membrane protein phosphorylation is associated with functional changes observed by chlorophyll fluorescence spectroscopy or chlorophyll fluorescence induction kinetics. Here we report on our recent protein sequence and other data concerning the identities of these phosphoproteins. We also discuss the significance of these findings for regulation by protein phosphorylation of photosynthesis in prokaryotes.     

Cloning of the hsp70 (dnaK) genes from Rhizobium meliloti and Pseudomonas cepacia: phylogenetic analyses of mitochondrial origin based on a highly conserved protein sequence.

The genes for hsp70 (or dnaK) have been cloned and sequenced from Rhizobium meliloti and Pseudomonas cepacia, two bacterial species belonging to the alpha- and beta-subdivisions of gram-negative proteobacteria, respectively. On the basis of global alignment of HSP70 proteins, several sequence signatures have been identified that are distinctive of mitochondrial homologs and gram-negative proteobacteria on the one hand and the chloroplasts and cyanobacteria on the other. Detailed phylogenetic analyses of HSP70 sequences from various eubacteria and eukaryotic organellar and cytosolic homologs support the inference regarding the origin of mitochondria from a member of the alpha-proteobacteria and of chloroplasts from cyanobacteria. The analysis presented here also suggests a monophyletic origin of the mitochondrial homologs.     

Occurrence of Hydrogenases in Cyanobacteria and Anoxygenic Photosynthetic Bacteria: Implications for the Phylogenetic Origin of Cyanobacterial and Algal Hydrogenases

Hydrogenases are important enzymes in the energy metabolism of microorganisms. Therefore, they are widespread in prokaryotes. We analyzed the occurrence of hydrogenases in cyanobacteria and deduced a FeFe-hydrogenase in three different heliobacterial strains. This allowed the first phylogenetic analysis of the hydrogenases of all five major groups of photosynthetic bacteria (heliobacteria, green nonsulfur bacteria, green sulfur bacteria, photosynthetic proteobacteria, and cyanobacteria). In the case of both hydrogenases found in cyanobacteria (uptake and bidirectional), the green nonsulfur bacterium Chloroflexus aurantiacus was found to be the closest ancestor. Apart from a close relation between the archaebacterial and the green sulfur bacterial sulfhydrogenase, we could not find any evidence for horizontal gene transfer. Therefore, it would be most parsimonious if a Chloroflexus-like bacterium was the ancestor of Chloroflexus aurantiacus and cyanobacteria. After having transmitted both hydrogenase genes vertically to the different cyanobacterial species, either no, one, or both enzymes were lost, thus producing the current distribution. Our data and the available data from the literature on the occurrence of cyanobacterial hydrogenases show that the cyanobacterial uptake hydrogenase is strictly linked to the occurrence of the nitrogenase. Nevertheless, we did identify a nitrogen-fixing Synechococcus strain without an uptake hydrogenase. Since we could not find genes of a FeFe-hydrogenase in any of the tested cyanobacteria, although strains performing anoxygenic photosynthesis were also included in the analysis, a cyanobacterial origin of the contemporary FeFe-hydrogenase of algal plastids seems unlikely. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Lauren Ancel Meyers]     

Assay for the transbilayer movement of polyisoprenoid-linked saccharides based on the transport of water-soluble analogues

Flippases are a class of membrane proteins that are proposed to facilitate the transbilayer movement of amphipathic polar lipids that are required for membrane biogenesis and the assembly of many diverse complex glycoconjugates in eukaryotic and prokaryotic cells. Despite their crucial roles in membrane biology, very little is known about their structures and the precise mechanism(s) by which they overcome the biophysical barriers of the hydrophobic core, and allow polar head groups to traverse membrane bilayers. This chapter presents methods based on the transport of water-soluble analogues that can be applied to investigate membrane proteins mediating the transverse diffusion of polyisoprenoid-linked glycolipid intermediates involved in the biosynthesis of N-linked glycoproteins, glycosylphosphatidylinositol anchors and bacterial polysaccharides.     

Constraints on the Biological Source(s) of the Orphan Branched Tetraether Membrane Lipids

A soil profile from the Saxnäs Mosse peat bog, Sweden, has been analysed for glycerol dialkyl glycerol tetraether (GDGT) membrane lipids and 16S rRNA genes in order to constrain the source of the yet ‘orphan,’ but supposedly bacterial, branched GDGTs. Branched GDGT lipids dominate over archaeal membrane lipids. The Acidobacteria comprise the dominant bacterial group, accounting for the majority of total Bacteria, and are generally more abundant than methanogenic archaea. Analysed acidobacterial strains did not contain branched GDGT lipids. Thus, the source organism must likely be searched for in other acidobacterial phyla or in another abundant group within the remaining bacteria.     

Crossing the lipid divide

Archaeal membrane lipids are structurally different from bacterial and eukaryotic membrane lipids, but little is known about the enzymes involved in their synthesis. In a recent study, Exterkate et al. identified and characterized a cardiolipin synthase from the archaeon Methanospirillum hungatei. This enzyme can synthesize archaeal, bacterial, and mixed archaeal/bacterial cardiolipin species from a wide variety of substrates, some of which are not even naturally occurring. This discovery could revolutionize synthetic lipid biology, being used to construct a variety of lipids with nonnatural head groups and mixed archaeal/bacterial hydrophobic chains.     

Detection of microbial biomass by intact polar membrane lipid analysis in the water column and surface sediments of the Black Sea

The stratified water column of the Black Sea produces a vertical succession of redox zones, stimulating microbial activity at the interfaces. Our study of intact polar membrane lipids (IPLs) in suspended particulate matter and sediments highlights their potential as biomarkers for assessing the taxonomic composition of live microbial biomass. Intact polar membrane lipids in oxic waters above the chemocline represent contributions of bacterial and eukaryotic photosynthetic algae, while anoxygenic phototrophic bacteria and sulfate-reducing bacteria comprise a substantial amount of microbial biomass in deeper suboxic and anoxic layers. Intact polar membrane lipids such as betaine lipids and glycosidic ceramides suggest unspecified anaerobic bacteria in the anoxic zone. Distributions of polar head groups and core lipids show planktonic archaea below the oxic zone; methanotrophic archaea are only a minor fraction of archaeal biomass in the anoxic zone, contrasting previous observations based on the apolar derivatives of archaeal lipids. Sediments contain algal and bacterial IPLs from the water column, but transport to the sediment is selective; bacterial and archaeal IPLs are also produced within the sediments. Intact polar membrane lipid distributions in the Black Sea are stratified in accordance with geochemical profiles and provide information on vertical successions of major microbial groups contributing to suspended biomass. This study vastly extends our knowledge of the distribution of complex microbial lipids in the ocean.     

The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.     

Vipp1: a very important protein in plastids?!

As a key feature in oxygenic photosynthesis, thylakoid membranes play an essential role in the physiology of plants, algae, and cyanobacteria. Despite their importance in the process of oxygenic photosynthesis, their biogenesis has remained a mystery to the present day. A decade ago, vesicle-inducing protein in plastids 1 (Vipp1) was described to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. Most follow-up studies clearly linked Vipp1 to membranes and Vipp1 interactions as well as the defects observed after Vipp1 depletion in chloroplasts and cyanobacteria indicate that Vipp1 directly binds to membranes, locally stabilizes bilayer structures, and thereby retains membrane integrity. Here current knowledge about the structure and function of Vipp1 is summarized with a special focus on its relationship to the bacterial phage shock protein A (PspA), as both proteins share a common origin and appear to have retained many similarities in structure and function.