Steal My Sunshine

Top row (l to r): Elysia chlorotica; spotted salamander adult; Lotharella globosa. Bottom row (l to r): diatom (Campylodiscus sp.); Paulinella chromatophora; diatom (Thalassiosira pseudonana). For credits, see end of article. Our world is swarming with symbioses. Sea anemones and clownfish, land plants and mycorrhizal fungi, rays and remora cleaner fish, corals and algae. All around us, radically different species team up in unconventional ways, forming long-lasting relationships that benefit both parties. Some species take these partnerships to the extreme, with one organism actually moving into the cells or tissues of another. Known as endosymbiosis, this type of interaction led to the creation of key organelles, including the mitochondrion, and has formed the basis of life as we know it. Among the more profitable endosymbioses is one that allows the host to derive energy from sunlight. The light-harvesting machines of plants and algae, for example, are the products of an ancient merger between a photosynthetic bacterium and a microbial eukaryote. Like mitochondria, present-day plastids—including both pigmented light-harvesting as well as unpigmented nonphotosynthetic chloroplasts—are fully and inescapably integrated into the host cell. Their journey from bacterium to internal solar-powered generator is responsible for much of the success and diversity of life on earth; no other cellular invention has had a greater impact on eukaryotic evolution. In fact, as scientists continue to uncover new and bizarre plastid-bearing lineages, it is becoming clear that many eukaryotic groups lacking plastids actually descend from photosynthetic ancestors. Some species are even in the process of generating novel plastid organelles. Catch me if you can The genesis of plastids follows a straightforward and generally agreed-upon plot: about 1.5 billion years ago, a heterotrophic eukaryote, which gained energy by consuming and digesting organic compounds, swallowed and retained a free-living photosynthetic cyanobacterium. Most biologists believe that this event, called a “primary” endosymbiosis, is the source of all known plastids. Genomic analyses of diverse members of land plants, green and red algae, and glaucophytes (tiny inconspicuous freshwater algae), which together form the eukaryotic supergroup Archaeplastida or Plantae, have demonstrated that the plastids within all archaeplastid lineages trace directly back to the original primary endosymbiotic event.1 The arrival and propagation of plastids within other eukaryotic supergroups, however, has followed a much more circuitous path, with plastids getting passed around and traded like playing cards. Once established in the Archaeplastida, plastids spread laterally to remote lineages through “secondary” endosymbioses, in which a nonphotosynthetic eukaryote engulfs and retains a eukaryote containing a primary plastid. (See illustration below.) Secondary plastids have popped up in the strangest places. For example, all known members of the eukaryotic supergroup Excavata are nonphotosynthetic with the exception of one lucky lineage of the euglenids, a phylum of ocean- and lake-dwelling unicells that like to snack on microbes, including green algae. This lineage, known as the euglenophytes, bagged a green algal plastid at some point in its evolutionary history, allowing its members to perform photosynthesis in addition to gathering food from their environment.2 A similar event occurred within tropical unicells called chlorarachniophytes. In this case, not only did the organisms acquire a photosynthetic plastid from a green alga, they also retained an extra eukaryotic nucleus, now called the nucleomorph. The nucleomorph still contains a functional genome, but it’s been whittled down from millions to a few hundred thousand nucleotides and may one day vanish completely. The journey from bacterium to internal solar-powered generator is responsible for much of the success and diversity of life on earth; no other cellular invention has had a greater impact on eukaryotic evolution. Opportunistic eukaryotes have also hijacked plastids from red algae. The number of times this has happened is debated, but it has undoubtedly had an immense impact on the planet’s biodiversity. It is estimated that more than half of all described microbial eukaryotes, including many algal members of phytoplankton, harbor red-algal-derived plastids. Red algal plastids are also found in dangerous human pathogens, such as the causative agents of malaria (Plasmodium) and toxoplasmosis (Toxoplasma). And like the green variety, red algae can also pass on their nuclei. Cryptophyte algae, unicellular organisms found in marine and freshwater environments, possess the plastid and nucleomorph of a secondary red algal endosymbiont. In a remarkable example of convergent evolution, the nucleomorph genomes of cryptophytes are strikingly similar in architecture to the green algae-derived nucleomorphs of chlorarachniophytes.2 PRIMARY ENDOSYMBIOSIS: About 1.5 billion years ago, a free-living photosynthetic cyanobacterium was engulfed by a heterotrophic eukaryote, which generated the plastids of all archaeplastid organisms—including land plants, green and red algae, and glaucophytes—studied to date.© THOM GRAVES SECONDARY ENDOSYMBIOSIS: “Secondary” endosymbioses occur when a nonphotosynthetic eukaryote swallows and retains a eukaryote, such as a green or red alga, which already contains a primary plastid. The secondary host will also sometimes retain the nucleus of the alga it engulfed.© THOM GRAVES “Tertiary” endosymbiosis, which occurs when a eukaryote takes up an alga containing a secondary plastid, complicates matters even further. A diversity of algal species, such as diatoms and cryptophytes, have donated their secondary plastids (with varying degrees of integration) to certain dinoflagellates. In many, or possibly all, of these cases, the dinoflagellate already had a secondary plastid to begin with, but discarded it for the new one. For instance, the spiral-swimming dinoflagellate Karenia, famous for causing massive “red tides” off the coast of Florida, swapped its original plastid for one from a haptophyte alga. One difficulty in tracking this dynamic exchange of plastids is that many species harboring plastids have lost their photosynthetic abilities, though they tend to retain the plastid that once mediated those processes.1 Green plants have ditched photosynthesis no less than 15 times, one example being the parasitic weed broomrape, a decimator of food crops worldwide. Numerous species with secondary plastids have also abandoned photosynthesis. Such an event is believed to have happened very early in the evolutionary history of apicomplexan protists, spawning an entire parasitic group of photosynthetic duds, which includes the malarial parasite Plasmodium. Recently, researchers discovered at the bottom of Sydney Harbor, Australia, the closest known photosynthetic relative of apicomplexan parasites.3 Called Chromera, this newly found alga is helping scientists piece together how apicomplexans first acquired their plastid and subsequently lost the ability to carry out photosynthesis.4 The fact that some species retain a plastid even after losing photosynthetic ability—the cellular equivalent of lugging around a broken light bulb—hints at additional roles for these acquired organelles. Indeed, research on nonphotosynthetic plants and algae has shown that their plastids still perform crucial steps in a number of cellular pathways, including fatty acid and heme biosynthesis. For example, despite feeding heterotrophically, Plasmodium cannot survive in the blood without its red-algal-derived plastid. Recently, scientists discovered that plastid-less Plasmodium can be rescued with isopentenyl pyrophosphate, a building block for important cellular lipids called isoprenoids. The finding suggests that during blood-stage growth the malaria plastid has a single, but essential, function: isoprenoid precursor synthesis, a pathway now being investigated as a potential target of new malaria drugs.5 Finally, although it is difficult to prove, certain lineages have probably completely lost photosynthetic stowaways acquired earlier in their evolution. Some of the best evidence for plastid extinction comes from the diarrhea-causing pathogen Cryptosporidium—cause of the infamous Milwaukee gastroenteritis outbreak of 1993, in which almost half a million people were infected and at least 69 died. Cryptosporidium’s ultrastructure and genome sequence reveal no signs of a plastid, but its phylogenetic position deep within the apicomplexan protists leaves little doubt that it descends from a plastid-bearing ancestor.1 The plastid-less heterokont Phytophthora, a nasty plant pathogen and cause of the Great Irish Potato Famine, also appears to have once contained a plastid, harboring genes apparently of red algal and cyanobacterial origin in its nuclear DNA. As more and more nuclear genomes are sequenced, it may turn out that some of the world’s shadiest protists once basked in the sunlight. COURTESY OF ROGER HANGARTER PHOTOSYNTHETIC WEIRDOS Some of the strangest creatures have managed to find themselves a photosynthetic companion, and they’ve done so in very creative ways. Here are a couple of the more bizarre examples of endosymbiosis, the study of which may lead to insights into the evolution of photosymbiosis. Thieving Slugs Elysia chlorotica is an inch-long, algae-sucking sea slug that hangs out in salt marshes along the east coast of North America, where it dines on the filamentous photosynthetic alga Vaucheria litorea. Using a horny, ribbonlike structure, Elysia punctures a hole in the Vaucheria cells, slurps up the alga’s plastids, sequesters them within its own digestive cells just beneath the epidermis, and feeds on the sugars they produce—a strategy fittingly dubbed kleptoplasty. Elysia can survive for months on the photosynthetic products of the stolen Vaucheria plastids. What is even more impressive, the plastids remain functional within Elysia for almost a year, after which the crafty slug feeds on some more Vaucheria to replenish the stock. If Elysia retains only the plastids of its algal prey, and does not maintain the algal nucleus, which contains most of the plastid-related genes, how do the Vaucheria plastids remain functional within the Elysia cells? The answer, researchers are learning, is that many Vaucheria genes are already integrated into Elysia DNA. Transcriptome sequencing suggests that more than 50 algal genes have been horizontally transferred to the Elysia genome, allowing the slug to operate the Vaucheria plastids and enjoy a solar-powered existence.1 Symbiotic Salamanders The spotted salamander Ambystoma maculatum is another algal connoisseur, but it prefers green algae, particularly Oophila amblystomatis, a unicellular freshwater flagellate closely related to the model organism Chlamydomonas. The symbiotic association between the salamander and green algae was described more than 120 years ago, but it wasn’t until last year that this relationship was proven to be an endosymbiotic one, making A. maculatum the first well-documented case of a vertebrate harboring a phototrophic organism in its cells. Using fluorescence and transmission electron microscopy as well as DNA sequencing, a team of Canadian and US researchers, led by Ryan Kerney of Dalhousie University in Halifax, Canada, showed that the embryonic tissues of the spotted salamander contain Oophila inside their cells.2 And although the alga appears to be degraded in adult salamanders, the researchers were able to amplify algal ribosomal RNA from adult reproductive tracts, suggesting that Oophila may be transmitted vertically from one Ambystoma generation to the next. Moreover, recent data suggest that there may be transfer of the alga’s photosynthetic products to its salamander host.3 The exact benefits of the relationship are unknown, but it is thought that the algae aid salamander embryo growth and hatchling survival, and that in return, Ambystoma supports the population growth of the algae. 1. S.K. Pierce et al., “Transcriptomic evidence for the expression of horizontally transferred algal nuclear genes in the photosynthetic sea slug, Elysia chlorotica,” Mol Biol Evol, 29:1545-56, 2012. 2. R. Kerney et al., “Intracellular invasion of green algae in a salamander host,” PNAS, 108:6497-502, 2011. 3. E.R. Graham et al., “Intracapsular algae provide fixed carbon to developing embryos of the salamander Ambystoma maculatum,” J Exp Biol, in press. VOLVOX CARTERICOURTESY OF AURORA NEDELCU Moving in The freewheeling history of plastid acquisition throughout the tree of life can make endosymbiosis seem easy, but hooking up with a photosynthetic partner, assimilating it into the workings of the host cell, and harnessing it for energy production is a long and complex process. A crucial step in keeping and controlling an endosymbiont is commandeering its DNA and controlling its proteins. After the primary endosymbiotic event, genes belonging to the internalized cyanobacterium began integrating into the host’s nuclear genome, or even disappearing completely.6 With each gene that migrated, the endosymbiont lost some autonomy and the host gained some control. As a result of this process, contemporary plastid genomes are puny, carrying only around 50–200 genes, whereas the host nucleus contains an enormous endosymbiotic “footprint” comprising between 5–20 percent of its genes. Remarkably, while most of the proteins encoded by these genes service the plastid, some have evolved nonplastid functions.6 For species that acquired a plastid secondarily by engulfing and retaining a photosynthetic eukaryote, there were additional waves of gene migration—this time from the nuclear genome of the algal endosymbiont carrying the plastid to the new host nuclear genome. Thus, taxa with secondary plastids, such as Plasmodium and the phytoplankton Thalassiosira, contain genes that began their journey in a cyanobacterium, migrated to the nuclear genome of an archaeplastid, and then moved again to the nuclear DNA of the secondary host. Organisms that have replaced their secondary plastid with a tertiary or different secondary one have even more complex genomes, in some cases harboring a mosaic of both red- and green-algal-derived genes. And then there are those algal lineages, the chlorarachniophytes and cryptophytes, that still retain the relic algal endosymbiont nuclear genome in a separate organelle (the nucleomorph), which is highly reduced in size, with many of those genes once carried by the primary host’s nucleus having migrated to the genome of the secondary host. Analyses of the complete host nuclear genomes of the chlorarachniophyte Bigelowiella natans and the cryptophyte Guillardia theta, which were recently sequenced by a group of scientists led by Bruce Curtis and John Archibald at Dalhousie University, Canada, 7 should reveal just how many genes have migrated from the nucleomorph to the new host genome. While the migration of endosymbiont genes to the host nucleus gives the host greater control over the plastid’s functions, it also creates a problem: the genes’ products must return to the plastid in order to do their job. For species with primary plastids, this has involved the installation of an intricate protein targeting and shuttling system. Decades of research have shown that nuclear-encoded plastid proteins have a unique tag, called a transit peptide, at the front end (N terminus) of their sequences that helps the cell direct them to the plastid, passing them through special import channels called translocons within the plastid membranes. Once inside, the tag is cleaved, and the protein can get to work. (See illustration below.) THE PLASTID GENE SHUFFLE View full size JPG | PDF© THOM GRAVESSpecies with secondary and tertiary plastids have built upon this system, but a few tweaks were needed. One of the consequences of being passed from eukaryote to eukaryote is the accumulation of membranes. Primary plastids have two membranes, both of which are cyanobacterial in origin, but secondary and tertiary plastids have three or four membranes. Shipping a protein into a triple-wrapped plastid is tricky. In all of the species for which it has been studied, such transport involves tacking on an additional tag, called a signal peptide, in front of the transit peptide. The signal peptide gets the nuclear-encoded plastid protein through the plastid’s outermost membrane, at which point it is clipped off to expose the transit peptide, thereby allowing the protein to pass through the remaining plastid membranes. Targeting proteins to the plastid is a key step in plastid integration, but exporting materials from the plastid is equally crucial. New findings from the genome of Cyanophora paradoxa, a unicellular glaucophyte alga, indicate that plastid-containing eukaryotes had help from an unexpected third party in learning how to transport the riches of photosynthesis from the plastid to the cytosol. When a team of international researchers, headed by Dana Price and Debashish Bhattacharya of Rutgers University, scanned the organism’s nuclear genome, they discovered sequences that were related to transporter genes in the bacterial parasites Chlamydia and Legionella, best known as the causes of chlamydia and Legionnaires’ disease, respectively.8 Turning to the nuclear genomes of red and green algae, the researchers uncovered some of these same genes. These results suggest that early in plastid evolution, genes from Chlamydia-like bacteria were horizontally transferred to eukaryotes and ultimately employed to help export photosynthetic products from the plastid to the cytosol. Clearly, the key to understanding plastid integration is in the genes. A window back in time Imagine what it would be like to go back in time a billion and a half years and watch the seminal events of the primary plastid integration unfold. Scientists are doing exactly this by studying the single-celled freshwater amoeba Paulinella chromatophora. Unlike a typical amoeba, P. chromatophora can perform photosynthesis, but it doesn’t have a plastid. It has a chromatophore, a pigment-containing, light-reflecting organelle. Like plastids, chromatophores descend from an endosymbiotic photosynthetic cyanobacterium. However, the endosymbiotic event that gave rise to the chromatophore occurred around 60 million years ago. Most species within the Paulinella genus do not have a chromatophore and survive by munching on microbes, which they catch using impressive phagocytic tentacles called filopods. But P. chromatophora is an exception: since capturing and incarcerating a cyanobacterium, this species has lost its feeding apparatus and gets by on photosynthesis alone. Just as in organisms carrying plastids, host cell division and chromatophore replication are synchronized, meaning that each daughter cell receives a chromatophore. Paulinella has been at the center of a long-standing debate as to whether the chromatophore is an endosymbiont or a full-fledged photosynthetic organelle, analogous to a plastid. Over the past 5 years, biologists have been sequencing genes within Paulinella’s chromatophore and nuclear genomes, and discovered that many of the genes that were originally present in the endosymbiont genome have disappeared, including some that were essential for maintaining a free-living existence. This means that the chromatophore is now permanently wedded to its host, but this alone is not enough to attain full-fledged organelle status. As proof that the assimilation has proceeded even further, at least 30 of the chromatophore’s genes have integrated into the host genome. PAULINELLA CHROMATOPHORACOURTESY OF EVA NOWACKThis past year, Eva Nowack and Arthur Grossman of the Carnegie Institution for Science discovered that three of these migrated genes do in fact code for chromatophore proteins.9 More importantly, these proteins are synthesized using the host’s cellular machinery and then shipped to the chromatophore, where they appear to be fully functional. This give-and-take relationship between the host and endosymbiont is believed by many to be the definitive step to becoming a genuine organelle, making P. chromatophora the only known example—outside the origin and spread of plastids—for which the shift from endosymbiont to photosynthetic organelle has occurred. Scientists are also studying the genome of the nonphotosynthetic Paulinella ovalis, which has neither a plastid nor a chromatophore. There they have identified at least two cyanobacterial-like genes, which were most likely transferred laterally to P. ovalis from its cyanobacterial prey.10 If true, this raises the question: Could horizontal gene transfer from cyanobacteria prior to endosymbiosis play a role in plastid integration? Perhaps P. chromatophora was pre-equipped to accept its chromatophore, aiding in its integration and retention. The answers to this and other questions are likely to be revealed through continued work on the Paulinella genus. For example, given P. chromatophora’s relatively new relationship with its photosynthetic organelle, it is unlikely that the protein-import apparatus of its chromatophore will prove as sophisticated as that of the plastid, which has had many hundreds of millions more years of fine-tuning. But comparing the architecture of these two systems, researchers should glean insights into the early stages of the formation of organelles. Other endosymbionts, including those yet to be discovered, will also inform the discussion. A quick scan of the tree of life reveals a plethora of taxa, from corals to fungi, that are partnered with photosynthetic species, and as biologists explore new lineages, they are sure to uncover even more examples of organisms engaging in this intimate interspecies relationship. David Smith is a Killam Postdoctoral Scholar in the Botany Department at the University of British Columbia, Vancouver, Canada. References 1. P.J. Keeling, “The endosymbiotic origin, diversification and fate of plastids,” Philos T R Soc B, 365:729-48, 2010. 2. J.M. Archibald, “The puzzle of plastid evolution,” Curr Biol, 19:R81-88, 2009. 3. R.B. Moore et al., “A photosynthetic alveolate closely related to apicomplexan parasites,” Nature, 451:959-63, 2008. 4. J. Janouškovec et al., “A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids,” PNAS, 107:10949-54, 2010. 5. E. Yeh, J.L. DeRisi, “Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum,” PLOS Biol, 9:e1001138, 2011. 6. T. Kleine et al., “DNA transfer from organelles to the nucleus: the idiosyncratic genetics of endosymbiosis,” Annu Rev Plant Biol, 60:115-38, 2009. 7. B.A. Curtis et al., “Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs,” Nature, doi:10.1038/nature11681, 2012. 8. D.C. Price et al., “Cyanophora paradoxa genome elucidates origin of photosynthesis in algae and plants,” Science, 335:843-47, 2012. 9. E.C.M. Nowack, A.R. Grossman, “Trafficking of protein into the recently established photosynthetic organelles of Paulinella chromatophora,” PNAS, 109:5340-45, 2012. 10. D. Bhattacharya et al., “Single cell genome analysis supports a link between phagotrophy and primary plastid endosymbiosis,” Sci Rep, 2:356, 2012. Photo credits from opening image. Top row (l to r): Pat Krug; Ryan Kerney; Yoshihisa Hirakawa. Bottom row (l to r): © Steve Gschmeissner/Science Source; Eva Nowack; © Biophoto Associates/Science Source

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