The role of horizontal gene transfer in kleptoplastidy and the ...

Commentary

commentary

Mobile Genetic Elements 3:2, e24773; March/April 2013; © 2013 Landes Bioscience

The role of horizontal gene transfer in kleptoplastidy and the establishment of photosynthesis in the eukaryotes Loïc Pillet Department of Genetics and Evolution; University of Geneva; Geneva, Switzerland

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Keywords: photosynthesis, endosymbiosis, photosymbiosis, kleptoplastidy, horizontal gene transfer, foraminifera, diatom, EST, plastid Submitted: 03/09/13 Accepted: 04/22/13 Citation: Pillet L. The role of horizontal gene transfer in kleptoplastidy and the establishment of photosynthesis in the eukaryotes; Mobile Genetic Elements 2013; 3:e24773; http://dx.doi.org/10.4161/ mge.24773 Correspondence to: Loïc Pillet; Email: [email protected] Commentary to: Pillet L, Pawlowski J. Transcriptome analysis of foraminiferan Elphidium margaritaceum questions the role of gene transfer in kleptoplastidy. Mol Biol Evol 2013; 30:66–9; PMID:22993235; http://dx.doi. org/10.1093/molbev/mss226

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ound in different eukaryotic lineages, kleptoplastidy is the ability to sequester chloroplasts from algal preys that are ingested and partially digested. While most of the genetic information required for the activity and maintenance of the kleptoplastids disappeared with the digestion of the algal nuclei, the photosynthetic organelles remain active during extended period of time. Many different hypotheses have been proposed to explain the longevity of the kleptoplastids within their host. The most popular one involves Horizontal Gene Transfer (HGT) from the algal genome to the host nucleus. In order to test this hypothesis, transcriptome-based analyses have been performed on different kleptoplastidic organisms during the past few years. However, the variability of the results obtained does not allow drawing a convincing conclusion regarding the precise role of HGT in kleptoplastidy. Understanding the mechanism that allow persistence of the plastids is crucial, not only for the characterization of kleptoplastidy, but also for important evolutionary questions surrounding endosymbiotic events and the emergence and spread of photosynthesis in the eukaryotes. Here, I discuss alternative theories that could explain the longevity of sequestered plastids in their host, with special focus on the simplest chloroplast stability hypothesis. The Emergence and Spread of Photosynthesis in the Eukaryotes The plastids found in today’s eukaryotes originated through the engulfment

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of ancestral free-living cyanobacteria approximately one billion years ago.1 After phagocytosis, these photosynthetic preys were usually digested in the food vacuoles, but exceptionally they could have been preserved as endosymbionts.2 During this process known as primary endosymbiosis, the symbionts were then stabilized into bona fide organelles (Fig. 1, #1), which resulted in the emergence of photosynthesis in the eukaryotes. Consecutive interactions between different eukaryotic lineage, such as secondary and tertiary endosymbioses (Fig. 1, #2), promoted the dispersal of photosynthesis and gave rise to the vast majority of photosynthetic eukaryotes found today.3 One of the main consequences of endosymbiosis was the flow of genetic information between the different cellular compartments that have newly been integrated. This Horizontal Gene Transfer (HGT), or more specifically Endosymbiotic Gene Transfer (EGT), was quantitatively important. In the case of primary endosymbiosis, EGT occurred from the cyanobacterial genome to the host nucleus, where hundreds of genes were incorporated. Consequently, eukaryotic plastid genomes usually encode less than 200 proteins3 of the predicted 1,000 to 5,000 that are required to sustain the chloroplast activity and maintenance.4,5 During subsequent secondary and tertiary endosymbiotic events, these genetic flows were even more important and had significant impact on the genomic structure of eukaryotic genomes. In these cases, genes were not only transferred from the endosymbiont plastid to the host nucleus, but also from the endosymbiont nucleus

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Figure 1. Different modes of dispersal of photosynthesis in the eukaryotes. Heterotrophic eukaryotes, represented in the center in light brown, became phototrophic using three main evolutionary strategies. The vast majority acquired their plastids through primary (#1) or secondary (#2) endosymbiosis (shown in brown). In that case, either a cyanobacteria, represented in blue, or a photosynthetic eukaryote, shown in purple, is engulfed and goes though important modifications, including massive Horizontal Gene Transfer (HGT), to become a primary (in blue) or secondary (in yellow) integrated organelle. Significant amount of heterotrophic eukaryotes developed photosymbiosis (shown in blue) as an alternative method to become phototrophic. Here, the algal symbiont, which can be either a cyanobacteria (#4), or any type of microalgae (#3), is ingested and preserved in the cytoplasm of the host without undergoing dramatic modifications. Another approach, called kleptoplastidy (#5) and shown in green, is used by several eukaryotic lineages to become photosynthetic. In this scenario, an algal prey (in purple) is ingested and while most of the cell is digested, the plastids (in yellow) are preserved and remain active during extended period of time.

(nucleomorph) to the host nucleus. The absence of primary algal nucleus in most of the organism containing secondary or tertiary-plastids attests the massive gene flow that occurred between endosymbiont and host nuclei. Alternatively, a number of heterotrophic eukaryotes acquired the capacity to perform photosynthesis bypassing the complex establishment of a permanent plastid through endosymbiosis. Indeed, these organisms rather retained intracellular photosynthetic ‘symbionts’, which could stand as a whole prokaryotic (Fig. 1, #4) or eukaryotic (Fig. 1, #3) cell, in photosymbiosis, or only as the chloroplasts of an algal prey in the case of kleptoplastidy (Fig. 1, #5). Photosymbiosis is found in many different lineages that are scattered in all eukaryotic supergroups and in certain of these groups it became a common way to acquire the photosynthetic capacity. For example, ciliates and dinoflagellates (Alveolata) significantly developed

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this strategy to use the sunlight energy,6,7 and lineages such as foraminiferans and radiolarians (Rhizaria) became real specialists of photosymbiotic associations with many different types of algae.8,9 Even though the acquisition of a permanent plastid through endosymbiosis is very complex, the establishment of a photosymbiotic association is certainly not trivial and a number of questions surround this process. An important one concerns the longevity of the symbiont in its host and the stability of such interaction. Although photosymbiotic associations are not permanent, they can last for extended periods of time. For example, Symbiodinium dinoflagellates can remain and multiply in their coral host during its all life.10 This observation also questions the degree of independence of symbionts within their host. Considering that photosymbionts still possess a full genome, they should be considered as autonomous.11 Indeed, culture experiments showed that they are.8


However, in the case of kleptoplastidy, the situation is much more complex. During this intriguing evolutionary process, the algal prey is engulfed by a heterotrophic eukaryote and the chloroplasts are sequestered while the rest of the cell is being digested. Despite the fact that the nuclear genome of the algae is not present anymore in the host cell, the stolen chloroplasts (kleptoplastids) stay functional for extended periods of time. Although kleptoplastidy remains marginal, this polyphyletic process is found in highly diverse eukaryotic lineages, including metazoans, ciliates, dinoflagellates and foraminifers. In Foraminifera, at least eight distinct genera have been reported to perform kleptoplastidy.12 However, the process was particularly well characterized for members of the genus Elphidium, which exclusively sequester plastids of diatom origin.13 As for many other organisms performing kleptoplastidy, the longevity of kleptoplastids within their host is remarkable. For instance, it has been shown that these organelles could remain active during several months after being ingested,14,15 suggesting that complex mechanisms are involved in their maintenance. The Role of Horizontal Gene Transfer In many points, the three main evolutionary processes that allowed the emergence and spread of photosynthesis in the eukaryotes significantly differ from each other (Fig. 1). An important issue is the degree of autonomy of the different ‘symbionts’ within their host, which is closely correlated with the structural modifications of the algal partner on the one hand, and to the process of Horizontal Gene Transfer (HGT) on the other hand. In the case of primary and secondary endosymbiosis (Fig. 1, #1 and #2), the algal partner went through important structural modifications to become an organelle fully integrated in the ‘host’ eukaryotic cell. Massive gene flow that took place between the different cellular compartments played a crucial role in the process of integration of the endosymbionts into bona fide organelles. One of the main consequences of this important HGT was the loss of the ‘endosymbionts’

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autonomy. Indeed, it is generally accepted that, unlike a symbiont, an organelle is not genetically independent anymore. Because of the large amount of genes that had been transferred to the host nucleus, the organelle directly relies on its host to maintain its genetic information.11 Previous studies suggested that any kind of interaction, such as trophic interaction or temporary association, could virtually lead to HGT.16,17 Therefore it is not surprising to find genes that have been transferred between photosymbiotic partners.18 However, while HGT is essential for the establishment of endosymbiosis, it should not be important in photosymbiotic associations (Fig. 1, #3 and #4). As a matter of fact, in that case the algal symbiont only goes through minimal modifications of its structure in order to adapt to the intracellular mode of life. However, the genetic integrity of the algal partner is not affected and it remains autonomous within the eukaryotic host cell. While the role of HGT has been well established in the cases of endosymbiosis and photosymbiosis, the significance of this process in kleptoplastidy (Fig. 1, #5) remains unclear and seems very variable from one eukaryotic lineage to another. Several transcriptomic studies carried on different models, like the sea slug Elysia chlorotica and the dinoflagellate Dinophysis acuminata, suggested that HGT between the algal prey and the host nuclei could explain, at least partially, the longevity of kleptoplastids in their hosts.19-21 However, similar experiments performed on other organisms, including other sea slug species and foraminiferan Elphidium margaritaceum, failed to recover transcriptionally active genes that could have been horizontally transferred and putatively implicated in the maintenance or activity of kleptoplastids.22,23 These contradictory results suggest that alternative hypotheses have to be considered and that HGT alone cannot explain the longevity of kleptoplastids, or at least not in the case of the foraminiferan E. margaritaceum. The Chloroplast Stability Hypothesis Many different hypotheses have already been discussed to explain the kleptoplastids


Figure 2. Chloroplast stability hypothesis. The longevity of kleptoplastids could be explained by this hypothesis. Different parameters (shown in blue) might contribute to the exceptional stability of the chloroplasts.

longevity, especially in the case of the sacoglossan sea slugs.22 These putative explanations included the possible retention of algal nuclei, which was shown to be true for the ciliate Myrionecta rubra performing karyoklepty.24 Nevertheless, this kind of process has never been observed in any other organism performing kleptoplastidy. Other scenarios included plastid genetic autonomy or dual targeting of proteins encoded in the host nucleus. Even though these hypotheses cannot straightforwardly be rejected, they both involve very complex mechanisms that have never been confirmed in any kleptoplastidic model, which makes them unlikely. For all these reasons, it seems that the most likely theory to explain the kleptoplastids longevity in Elphidium is the chloroplast stability hypothesis. Despite the fact that this model represents the simplest explanation, it has never been considered as serious as the HGT hypothesis. However, multiple observations by different authors are congruent with this theory. First, using different organisms, various studies tried to precisely establish the longevity of the kleptoplastids within their host. Conclusions were quite different from one model to another, varying from few days25 to several months.14,15 However, all the analyses suggested that the turnover of kleptoplastids was higher when the host was incubated in light/dark cycles than in permanent dark conditions.26,27 This is interesting as it shows that the


longevity of kleptoplastids is directly correlated with environmental conditions. The more the plastid machinery is under demanding conditions, the higher its turnover is. Even though this observation seems obvious, it reveals the lack of an efficient maintenance mechanism, which is congruent with the chloroplast stability hypothesis. Indeed, this theory suggests that the central reason why kleptoplastids remain functional during extended period of time is their exceptional stability. Undoubtedly, several parameters contribute to the plastids stability (Fig. 2). An important one is the very nature of the organelle. Previous analyses showed that different chloroplasts extracted from different algal sources and incubated in similar conditions had distinct longevities.28 Therefore, the choice of the chloroplast donor is probably not random and mechanisms allowing the recognition of specific algal strains might be involved in the longevity of kleptoplastids. Certainly, HGT represents another important parameter that significantly contributes to the chloroplast stability in certain organisms. By allowing the renewal of key proteins involved in the photosynthetic process, HGT could prolong the activity of plastids in a significant way. However, it should not be considered as the central reason for the longevity of kleptoplastids. Another interesting approach to decrease the turnover of kleptoplastids has been described by some authors and involves behavioral

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adaptation of the host organism. In order to preserve the plastids they sequestered from high sun-exposure, some sacoglossan mollusk species developed a specific shading behavior, using their parapodial flaps to protect kleptoplastids from direct light exposure.19 Similarly, the habitat might represent an important factor for the longevity of plastids in their host. Indeed, it has been shown that kleptoplastids turnover dramatically decreases in organisms living in specific habitats where the level of irradiance is very low. This is the case of the deep-sea foraminiferan species Nonionella stella that can maintain its kleptoplastids for up to one year.15 In the same way, it cannot be excluded that certain kleptoplastidic organisms adapt their habitat in order to preserve the plastids they sequester. Many other parameters, such as the host intracellular physico-chemical composition, might influence the stability of the kleptoplastids. Additionally, it is important to notice that there is probably no common strategy that allows explaining the longevity of chloroplasts in all the different kleptoplastidic organisms that are scattered among the whole eukaryotic radiation. Every single species probably uses the most adapted strategy depending on specific parameters, such as its cellular structure, habitat or ecology. Furthermore

each strategy certainly involves a combination of different approaches dedicated to the maintenance of kleptoplastids. This is congruent with the fact that transcriptomic analyses gave rise to completely different conclusions when trying to test the HGT hypothesis. The sea slug findings represent a good example, with the species E. chlorotica showing reasonable amount of transcriptionally active genes that have been transferred from the algal genome to the host nucleus,19 while no HGT evidence could be found in E. timida, another species belonging to the same genus that is known for its shading behavior.19 More than unraveling a curious process of itself, the understanding of the mechanism leading to plastids longevity will impact the correct interpretation of kleptoplastidy in an evolutionary context. Based on HGT evidences, different authors previously described this process as an intermediate step between photosymbiosis and endosymbiosis.29,30 However, if the chloroplast stability hypothesis is correct, it might be more appropriate to consider kleptoplastidy as a kind of plastids farming, as previously suggested.31,32 Ecological significance of photosynthesis is obvious and eukaryotes have developed a variety of strategies to acquire the photosynthetic capacity. From permanent

References

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1. Yoon HS, Hackett JD, Ciniglia C, Pinto G, Bhattacharya D. A molecular timeline for the origin of photosynthetic eukaryotes. Mol Biol Evol 2004; 21:809-18; PMID:14963099; http://dx.doi. org/10.1093/molbev/msh075 2. Reyes-Prieto A, Weber AP, Bhattacharya D. The origin and establishment of the plastid in algae and plants. Annu Rev Genet 2007; 41:147-68; PMID:17600460 ; http://dx.doi.org/10.1146/ annurev.genet.41.110306.130134 3. Archibald JM. The puzzle of plastid evolution. Curr Biol 2009; 19:R81-8; PMID:19174147; http:// dx.doi.org/10.1016/j.cub.2008.11.067 4. Martin W, Rujan T, Richly E, Hansen A, Cornelsen S, Lins T, et al. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc Natl Acad Sci U S A 2002; 99:12246-51; PMID:12218172; http://dx.doi. org/10.1073/pnas.182432999 5. Richly E, Leister D. An improved prediction of chloroplast proteins reveals diversities and commonalities in the chloroplast proteomes of Arabidopsis and rice. Gene 2004; 329:11-6; PMID:15033524; http:// dx.doi.org/10.1016/j.gene.2004.01.008 6. Dolan J. Mixotrophy in Ciliates: A Review of Chlorella Symbiosis and Chloroplast Retention. Mar Microb Food Webs 1992; 6:115-32

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plastid acquisition through endosymbiosis, to chloroplast sequestration or photosymbiosis, heterotrophic eukaryotes developed various evolutionary approaches to capture the sunlight energy. Regardless of the fundamental differences that exist between these strategies they also have much in common. For that reason, studying separately the different approaches that were used to spread photosynthesis in the eukaryotes is essential to answer many important questions concerning the evolutionary history of eukaryotes. More than the understanding of this fascinating process itself, studying kleptoplastidy could bring new insights into the establishment of complex plastid-targeting systems, into the reasons to maintain a nucleomorph or into any of these transitory-looking evolutionary curiosities surrounding the emergence of photosynthesis in the eukaryotes. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

I would like to thank Jan Pawlowski and Roberto Sierra for their comments and very useful discussions. This work was supported by the Swiss National Science Foundation (grant number 31003A_140766) and by a G. and A. Claraz Donation. 15. Grzymski J, Schofield OM, Falkowski PG, Bernhard JM. The function of plastids in the deep-sea benthic foraminifer, Nonionella stella. Limnol Oceanogr 2002; 47:1569-80; http://dx.doi.org/10.4319/ lo.2002.47.6.1569 16. Hehemann JH, Correc G, Barbeyron T, Helbert W, Czjzek M, Michel G. Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature 2010; 464:908-12; PMID:20376150; http://dx.doi.org/10.1038/nature08937 17. Keeling PJ, Palmer JD. Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet 2008; 9:60518; PMID:18591983; http://dx.doi.org/10.1038/ nrg2386 18. Starcevic A, Akthar S, Dunlap WC, Shick JM, Hranueli D, Cullum J, et al. Enzymes of the shikimic acid pathway encoded in the genome of a basal metazoan, Nematostella vectensis, have microbial origins. Proc Natl Acad Sci U S A 2008; 105:25337; PMID:18268342; http://dx.doi.org/10.1073/ pnas.0707388105 19. Pierce SK, Fang X, Schwartz JA, Jiang X, Zhao W, Curtis NE, et al. Transcriptomic evidence for the expression of horizontally transferred algal nuclear genes in the photosynthetic sea slug, Elysia chlorotica. Mol Biol Evol 2012; 29:1545-56; PMID:22319135; http://dx.doi.org/10.1093/molbev/msr316

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20. Rumpho ME, Worful JM, Lee J, Kannan K, Tyler MS, Bhattacharya D, et al. Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica. Proc Natl Acad Sci U S A 2008; 105:17867-71; PMID:19004808; http:// dx.doi.org/10.1073/pnas.0804968105 21. Wisecaver JH, Hackett JD. Transcriptome analysis reveals nuclear-encoded proteins for the maintenance of temporary plastids in the dinoflagellate Dinophysis acuminata. BMC Genomics 2010; 11:366; PMID:20537123; http://dx.doi.org/10.1186/14712164-11-366 22. Pelletreau KN, Bhattacharya D, Price DC, Worful JM, Moustafa A, Rumpho ME. Sea slug kleptoplasty and plastid maintenance in a metazoan. Plant Physiol 2011; 155:1561-5; PMID:21346171; http://dx.doi. org/10.1104/pp.111.174078 23. Pillet L, Pawlowski J. Transcriptome analysis of foraminiferan Elphidium margaritaceum questions the role of gene transfer in kleptoplastidy. Mol Biol Evol 2013; 30:66-9; PMID:22993235; http:// dx.doi.org/10.1093/molbev/mss226 24. Johnson MD, Oldach D, Delwiche CF, Stoecker DK. Retention of transcriptionally active cryptophyte nuclei by the ciliate Myrionecta rubra. Nature 2007; 445:426-8; PMID:17251979; http://dx.doi. org/10.1038/nature05496


25. Minnhagen S, Carvalho WF, Salomon PS, Janson S. Chloroplast DNA content in Dinophysis (Dinophyceae) from different cell cycle stages is consistent with kleptoplasty. Environ Microbiol 2008; 10:2411-7; PMID:18518896; http://dx.doi. org/10.1111/j.1462-2920.2008.01666.x 26. Hinde R, Smith DC. The role of photosynthesis in the nutrition of the mollusc Elysia viridis. Biol J Linn Soc Lond 1975; 7:161-71; http://dx.doi. org/10.1111/j.1095-8312.1975.tb00738.x 27. Lopez E. Algal Chloroplasts in the Protoplasm of Three Species of Benthic Foraminifera: Taxonomic Affinity, Viability and Persistence. Mar Biol 1979; 53:201-11; http://dx.doi.org/10.1007/BF00952427 28. Green BJ, Fox TC, Rumpho ME. Stability of isolated algal chloroplasts that participate in a unique mollusc/ kleptoplast association. Symbiosis 2005; 40:31-40 29. Gast RJ, Moran DM, Dennett MR, Caron DA. Kleptoplasty in an Antarctic dinoflagellate: caught in evolutionary transition? Environ Microbiol 2007; 9:39-45; PMID:17227410; http://dx.doi. org/10.1111/j.1462-2920.2006.01109.x


30. Johnson MD. The acquisition of phototrophy: adaptive strategies of hosting endosymbionts and organelles. Photosynth Res 2011; 107:117-32; PMID:20405214 ; http://dx.doi.org/10.1007/ s11120-010-9546-8 31. Leutenegger S. Symbiosis in Benthic Foraminifera: Specificity and Host Adaptations. J Foraminiferal Res 1984; 14:16-35; http://dx.doi.org/10.2113/ gsjfr.14.1.16 32. Nowack ECM, Melkonian M. Endosymbiotic associations within protists. Philos Trans R Soc Lond B Biol Sci 2010; 365:699-712; PMID:20124339; http://dx.doi.org/10.1098/rstb.2009.0188

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