Metabolic connectivity as a driver of host and endosymbiont integration Slim Karkara,1, Fabio Facchinellib,1, Dana C. Pricea, Andreas P. M. Weberb,2, and Debashish Bhattacharyaa,2 aDepartment of Ecology, Evolution, and Natural Resources, Rutgers University, New Brunswick, NJ 08901; and bInstitut für Biochemie der Pflanzen, Cluster of Excellence on Plant Sciences, Heinrich-Heine Universität, D-40225 Düsseldorf, Germany Edited by Patrick J. Keeling, University of British Columbia, Vancouver, Canada, and accepted by the Editorial Board March 6, 2015 (received for review December 19, 2014) The origin of oxygenic photosynthesis in the Archaeplastida com- Given the fundamental role of algae and plants as primary mon ancestor was foundational for the evolution of multicel- producers in aquatic and terrestrial habitats (17, 18), much at- lular life. It is very likely that the primary endosymbiosis that tention has focused on elucidating the rules that underlie pri- explains plastid origin relied initially on the establishment of a mary plastid origin in Archaeplastida and, more recently, in metabolic connection between the host cell and captured cyano- Paulinella. We have previously made the argument that a key, bacterium. We posit that these connections were derived primarily and likely fundamental, step in endosymbiont integration (i.e., from existing host-derived components. To test this idea, we used enslavement) was linking the metabolism of the host and endo- phylogenomic and network analysis to infer the phylogenetic symbiont, thereby allowing regulatory pathways to evolve that origin and evolutionary history of 37 validated plastid innermost would maximize connectivity of the partners, and as a result, host membrane (permeome) metabolite transporters from the model fitness (19–21). The major players in this process are trans- plant Arabidopsis thaliana. Our results show that 57% of these porters located in the innermost envelope membrane of plastids transporter genes are of eukaryotic origin and that the captured (the plastid envelope permeome) that are responsible for the cyanobacterium made a relatively minor (albeit important) con- controlled movement of metabolites to and from the endosym- tribution to the process. We also tested the hypothesis that the biont (e.g., energy as photosynthetically fixed carbon; the pre- bacterium-derived hexose-phosphate transporter UhpC might sumed raison d’être for plastid origin). Our previous work showed have been the primordial sugar transporter in the Archaeplastida that members of the nucleotide sugar transporter family [NST; ancestor. Bioinformatic and protein localization studies demon- within the drug/metabolite superfamily (DMS)] gave rise through strate that this protein in the extremophilic red algae Galdieria gene duplication and divergence to a variety of plastidic sugar sulphuraria and Cyanidioschyzon merolae are plastid targeted. transporters in red algae and Viridiplantae (Fig. S1) (19, 22, Given this protein is also localized in plastids in the glaucophyte 23). These genes encode the plastidic phosphate translocators alga Cyanophora paradoxa, we suggest it played a crucial role in (pPTs) that facilitate the strict counter exchange of a host-derived early plastid endosymbiosis by connecting the endosymbiont and inorganic orthophosphate (Pi) for an endosymbiont-derived phos- host carbon storage networks. In summary, our work significantly phorylated C3, C5, or C6 carbon compound (e.g., triose phosphate, advances understanding of plastid integration and favors a host- xylulose-5-phosphate, glucose-6-phosphate). Along with the shared centric view of endosymbiosis. Under this view, nuclear genes of ancestry of the plastid protein import system (6, 24), this in- either eukaryotic or bacterial (noncyanobacterial) origin provided novation provides one of the strongest pieces of evidence key elements of the toolkit needed for establishing metabolic con- that two major members of the Archaeplastida (red algae and nections in the primordial Archaeplastida lineage. Viridiplantae) are monophyletic. The tree also shows that members of the “chromalveolates” (e.g., stramenopiles, apicomplexans, Arabidopsis thaliana | endosymbiosis | evolution | network analysis | cryptophytes) gained their pPT homologs through red algal symbiont integration endosymbiosis. The retention of hexose phosphate transport as the primary carbon export mechanism in the third arm of the he origin and establishment of the photosynthetic organelle, Archaeplastida, the Glaucophyta (6), provides another in- Tthe plastid, is heralded as one of the most important bi- triguing twist in the story of primary endosymbiosis and will be ological innovations on our planet (1, 2). This primary endosym- discussed in detail below. This transporter (UhpC) originated biosis occurred more than a billion years ago and resulted from through horizontal gene transfer (HGT) from a bacterial the engulfment and enslavement of a once free-living cyanobac- source. The work on pPTs inspired us to look in more detail terium by a phagotrophic protist (3). Primary plastid capture into the evolutionary history and functional diversification putatively occurred a single time in the common ancestor of the eukaryotic supergroup Archaeplastida (also known as Plantae) This paper results from the Arthur M. Sackler Colloquium of the National Academy of that comprises the green algae and land plants (Viridiplantae), Sciences, “Symbioses Becoming Permanent: The Origins and Evolutionary Trajectories of red algae, and glaucophyte algae (4–6). Once established in these Organelles,” held October 15–17, 2014, at the Arnold and Mabel Beckman Center of the lineages, the plastid spread to other lineages such as diatoms, National Academies of Sciences and Engineering in Irvine, CA. The complete program and video recordings of most presentations are available on the NAS website at www. haptophytes, most dinoflagellates, and euglenids, through red or nasonline.org/Symbioses. green algal secondary endosymbiosis, and in some dinoflagellates, Author contributions: A.P.M.W. and D.B. designed research; S.K., F.F., D.C.P., and D.B. through tertiary endosymbiosis of a secondary endosymbiont- performed research; S.K. and F.F. contributed new reagents/analytic tools; S.K., D.C.P., containing alga (7, 8). The exceptional rarity of primary plastid and D.B. analyzed data; and F.F., A.P.M.W., and D.B. wrote the paper. endosymbiosis is supported by there being only one other known The authors declare no conflict of interest. case of a cyanobacterium-derived photosynthetic organelle (9). This article is a PNAS Direct Submission. P.J.K. is a guest editor invited by the Editorial “ ” Board. This chromatophore is found in a single lineage of photosyn- 1 Paulinella chromatophora S.K. and F.F. contributed equally to this work. thetic filose amoebae that includes and 2 – To whom correspondence may be addressed. Email: [email protected] its sister taxa (10 14). This independent primary endosymbiosis or [email protected]. ∼ likely occurred 60 Mya, and the plastid donor was a member of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the α-cyanobacterium clade (15, 16). 1073/pnas.1421375112/-/DCSupplemental. 10208–10215 | PNAS | August 18, 2015 | vol. 112 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1421375112 Downloaded by guest on October 4, 2021 PAPER of other plastid-targeted transporters and here we present an existing host-derived proteins to the plastid envelope permeome COLLOQUIUM analysis of these proteins in Archaeplastida. Our approach rather than by the wholesale repurposing of endosymbiont genes was to use phylogenomic and protein similarity network (22, 25, 29). The genes encoding these ancient transporters analysis of the validated plastidic transporters from Arabi- presumably underwent duplication(s) with one or more copies dopsis thaliana to deduce their evolutionary histories and or- taking on plastid-specific functions (Figs. S1 and S2). This host- igins (25). We also studied the phylogeny and cellular centric perspective has also been taken to suggest that the eu- localization of UhpC proteins in red algae to gain insights into karyote rather than the endosymbiont was the major contributor what may have been the ancestral pathway of sugar transport to protein sorting components with the endosymbiont outer in Archaeplastida. These data, combined with recent evidence membrane being the initial target for integration (30, 31). A of apparent translocon-independent protein import to the contrasting view (32) relies on genetic tinkering with endosym- photosynthetic organelle in Paulinella (26), provide a novel biont genes to derive basic components of mitochondrial trans- perspective on endosymbiont integration. Based on these data, locons (31). This lively discussion is far from settled, but it is we suggest that metabolic connectivity, whereby recruitment of clear that distinguishing between these hypotheses with regard to existing host-derived transporters to the plastid innermost mem- different endosymbiont traits depends not only on identifying the brane, was likely an early and fundamental step in unlocking the putative genetic toolkit for endosymbiont integration (with sol- metabolic potential of the captured cyanobacterium. ute transport and protein import being obvious candidates) but equally importantly, on elucidating their phylogenetic history. Results and Discussion Whereas explaining the origins