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Home , Amphibacillus, Amphibacillus xylanus, Caldalkalibacillus, Chlamydiae, Chloroflexi (phylum), Cyanidioschyzon

Metabolic connectivity as a driver of and integration

Slim Karkara,1, Fabio Facchinellib,1, Dana C. Pricea, Andreas P. M. Weberb,2, and Debashish Bhattacharyaa,2

aDepartment of Ecology, , and Natural Resources, Rutgers University, New Brunswick, NJ 08901; and bInstitut für Biochemie der Pflanzen, Cluster of Excellence on 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 in the com- Given the fundamental role of and as primary mon ancestor was foundational for the evolution of multicel- producers in aquatic and terrestrial habitats (17, 18), much at- lular . It is very likely that the primary endosymbiosis that tention has focused on elucidating the rules that underlie pri- explains origin relied initially on the establishment of a mary plastid origin in Archaeplastida and, more recently, in metabolic connection between the host 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 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 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 localization studies demon- within the drug/metabolite superfamily (DMS)] gave rise through strate that this protein in the extremophilic gene duplication and divergence to a variety of plastidic sugar sulphuraria and merolae are plastid targeted. transporters in red algae and Viridiplantae (Fig. S1) (19, 22, Given this protein is also localized in plastids in the 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 , 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 (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 (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 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 ,” held October 15–17, 2014, at the Arnold and Mabel Beckman Center of the lineages, the plastid spread to other lineages such as , 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. , most , 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 (15, 16). 1073/pnas.1421375112/-/DCSupplemental.

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of other plastid-targeted transporters and here we present an existing host-derived 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 of plastid protein translocon com- Phylogenomic Analysis of Arabidopsis Plastidic Transporters. Phylo- ponents still remain a challenge (33), here we are able to provide genomic analysis of the 34 Arabidopsis thaliana plastid envelope strong evidence for a host-dominated process with regard to plastid transporters listed in ref. 25 and 3 others that were more recently metabolite transport. This hypothesis is buttressed by the fact that Paulinella described in this [nitrite transporters At5g62720 (AtNITR2;1) permanent (e.g., the chromatophore ge- and At3g47980 (AtNITR2;2), and the glycolate/glycerate trans- nome) are characterized by massive decay through outright – porter At1g32080 (PLGG1)] (27, 28) were used as queries against loss or endosymbiotic gene transfer (EGT) (12 14). Therefore, a comprehensive local genome database (for details, see Methods innovations relating to the compartment are more likely to origi- and Table S1). The resulting alignments and trees, using either nate in the gene/function-rich nuclear host genome or via HGT to the host from foreign sources. the full alignment length or a gap-trimmed version (available for EVOLUTION download at cyanophora.rutgers.edu/transporters/) were inspected Network Analysis of Arabidopsis Plastidic Transporters. To gain a to determine the phylogenetic origins of the plant transporter phylogeny-independent perspective on plastid transporter evo- families. Most of these Viridiplantae proteins were found to be lution, we generated protein similarity networks with an all vs. all either nested with strong bootstrap support (generally >90%; BLASTP analysis of the nonredundant list of database hits to the Table S1) within a variety of eukaryotic lineages or to be asso- Arabidopsis Methods Arabidopsis 34 transporters described in ref. 25 (see for ciated with . A total of 57% of plastidic details). Using a 70% query coverage cutoff resulted in these transporters were of host (ancient eukaryotic; nine families) data forming 16 major (i.e., containing many members) connected origin, only 8% (three families) were of cyanobacterial (putative components (gene families) that together include all of the endosymbiont) origin, 24% were derived from noncyanobacterial transporter families, as well as 5 minor components that include prokaryotes (presumably many via HGT) with four families pu- highly diverged family members (e.g., CLT2, PIC1; Fig. 2). tatively derived from [i.e., PHT2.1, (NTT1, NTT2), Placement of the major components (boxed) into transporter su- (DiT1, DiT2.1, DiT2.2), HMA1], and a small number were either perfamilies using the Transporter Classification Database (TCDB; plant-specific or of uncertain affiliation (Fig. 1). The divergent www.tcdb.org/) shows that most plastidic transporters are an- origin of some transporter families is exemplified by the phy- ciently diverged and derive from distinct superfamilies, with six logeny of plastidic phosphate transporters (PHTs) shown in components (boxed in gray field) comprised solely of eukaryotic or Fig. S2. plant-specific sequences (Fig. 2). Nonetheless, several interesting These results (and previous work) support the scenario that components were identified, including the expected pPT family the current, and presumably the primordial, contribution to (e.g., PPT, TPT, XPT) that is derived from a single gene du- plastid metabolite transport was dominated by the retargeting of plication of an existing NST in the ancestor of red algae and Viridiplantae (Fig. S1). Another family of interest contained the copper transporting P-type ATPases HMA1 and PAA1 (34–36). This component included the eukaryote derived ACA1/PEA1 (a calcium ATPase; Table S1) family that shares Total number of genes: Prokaryotic [8 families]: Host (eukaryote) some links with the HMA1/PAA1 cluster (Figs. 2 and 3). To 24% Permease (Pic1) 57% Copper (PAA1) Other prokaryotes test this latter connection, we imposed a minimum pairwise Plant specific Folate (FT) protein identity threshold of 40%. This restriction was applied 8% Unclear Phosphate (PHT2.1) e ≤ −5 Dicarboxylate (Dit1, 2.1, 2.2) together with the standard -value cutoff 10 and a minimum Host (eukaryote) derived [9 families]: Copper (HMA1) of 70% query hit coverage to produce a new network. Under Sugar/phosphate (TPT, PPTs, GPTs, XPT) Nucleotide (NTT1, 2) this stringent condition (Fig. S3), the ACA1/PEA1 family be- Phosphate (PHT4.2-4.5) Nitrite (AtNITR2;1, 2;2) came an independent component, whereas links remained be- MCF (SAMT/C, BT1, NDT1) Calcium ATPase (ACA1/PEA1) Plant-specific [2 families]: tween many of the copper transporters. Inspection of the Nitrate (Nitr1) Stress response (CLT1-3) protein alignment suggested that the links to ACA1/PEA1 were 8% Folate (FLT1) Glycolate/glycerate (PLGG1) likely explained by the presence of two shared domains that Glucose (pGlcT) Unclear [2 families]: resulted in the network interaction (Fig. 3A). Magnesium (MGT10) Bile acid (BAT5) Labeling this network in two different ways (Fig. 3 A and B) Cationic proton (CHX23) Maltose (Mex1) shows that both of the copper transporters in Arabidopsis and in Fig. 1. Results of phylogenomic analysis of A. thaliana plastid-targeted other are derived from prokaryotic ancestors. Phy- transporters. The specific contributions and their numbers made by the eu- logenetic analysis of the combined alignment resolves gene ori- karyote host (brown text), cyanobacteria (blue text), prokaryotes other than gins with PAA1 having a cyanobacterial (endosymbiont) provenance, cyanobacteria (gray text), and other sources are shown. whereas HMA1 has a chlamydial origin (Fig. 3C). Intriguingly,

Karkar et al. PNAS | August 18, 2015 | vol. 112 | no. 33 | 10209 Downloaded by guest on October 4, 2021 MFSMFS POT/PTR DMT CLT3 FT MC CLT1 CLT2 Nitr1 PIC1

Arabidopsis query MC MITMIT CHX23 PHT2.1 Other eukaryotes Chlamydiae Cyanobacteria FOLT1 CPA2A2 MGT10 Other prokaryotes

DiT1 PHT2.1 MC NDT1 Mex1

SAMT/C PiT DiT2.2 DASS MEX BT1 DiT2.1 BASS PPT1 DMTDMT PHT4.2 CLT2 PHT4.3 PHT4.4 PPT2 GPT2 PHT4.5 GPT1 BAT5 TPT XPT pGlcT MFSMFS P-typepe ATPasATPasee ACA1/PEA1 CLT2 HMA1 FT NTT1 PIC1 PAA1 CLT1 AAA NTT2 2.A.12 ATP:ADP Antiporter (AAA) Family 2.A.28 Bile Acid:Na Symporter (BASS) Family 2.A.37 Monovalent Cation:Proton Antiporter-2 (CPA2) Family 2.A.47 Divalent Anion:Na+ Symporter (DASS) Family 2.A.7 Drug/Metabolite Transporter (DMT) Superfamily 1.A.35 CorA Metal Ion Transporter (MIT) Family 2.A.29 Mitochondrial Carrier (MC) Family 2.A.84 Maltose Exporter (MEX) Family 2.A.1 Major Facilitator Superfamily (MFS) 2.A.20 Inorganic Phosphate Transporter (PiT) Family 3.A.3 P-type ATPase (P-ATPase) Superfamily 2.A.17 Proton-dependent Oligopeptide Transporter (POT/PTR) Family

Fig. 2. Network analysis of 34 Arabidopsis plastid-targeted transporters. Each connected component is identified with respect to transport function, superfamily classification, and the taxonomic composition of network nodes. Highly diverged family members that form independent components under the cutoff used for pairwise comparison (i.e., 70% coverage) are also shown. Components comprised solely of eukaryotic sequences are shown in the gray boxes.

both of these prokaryotes are implicated in plastid origin with the cytosol; Figs. S4 and S5]. These three adenosine-based Chlamydiae, providing some key genes required for starch syn- transporters are of eukaryotic origin and have evolved differing thesis and metabolic integration of the endosymbiont (37). The transport activities during Archaeplastida evolution. Phyloge- biological meaning of this network result is, however, unclear. netic analysis of the combined alignment (Fig. S4C) shows that Copper is an essential micronutrient that is a component of all of these plastid (re)targeted transporter families likely trace (cofactor of plastocyanin) and Cu/Zn superoxide their origin to a single gene that was present in the common dismutase that is involved in the dismutation of superoxide to ancestor of eukaryotes. This hypothesis is supported by the ab- hydrogen peroxide (38). However, due to its high toxicity (e.g., it sence of prokaryotic homologs of this gene family in the tree can catalyze the production of free radicals), in plant cells, (i.e., using our search parameters) and the observation that the copper is associated with Cu chaperones (39, 40). Recent work transporter subtrees contain a wide array of eukaryotic lineages suggests that the HMA1 and PAA1 transporters operate as dis- (e.g., fungi, metazoans, red algae), suggesting ancient prove- tinct pathways for copper import into plastids (36), thereby pu- tatively explaining the maintenance of two diverged copies in nance in the ancestor of these taxa. Archaeplastida. In summary, the phylogenomic and network analyses point to Another intriguing connection uncovered by the network a fundamental role for the host cell in the evolution of metabolic analysis was between three members of the mitochondrial connectivity of the endosymbiont. This development is not only carrier superfamily (MCF) that includes the SAMT/C [coun- in terms of the number of proteins that have been recruited to ter exchange of S-adenosylmethionine (SAMT; cytosol) with the envelope permeome but also in the crucial roles they play, S-adenosylhomocysteine (SAHC; plastid)], NDT1 [counter exchange from fixed carbon transport to the delivery of methyl donors + of NAD (cytosol) with AMP or ADP (plastid)], and BT1 (in (SAMT/C) to the plastid to facilitate prenyllipid biogenesis or to Arabidopsis, unidirectional flow of plastid AMP, ADP, ATP to regulate the synthesis of aspartate-derived amino acids (41).

10210 | www.pnas.org/cgi/doi/10.1073/pnas.1421375112 Karkar et al. Downloaded by guest on October 4, 2021 PAPER COLLOQUIUM

ACA1/PEA1: calcium ATPase AB(At1g27770) ACA1/PEA1 Arabidopsis query Other eukaryotes Chlamydiae Cyanobacteria Other prokaryotes

PAA1: copper P-type ATPase PAA1 (At4g33520) E1-E2_ATPase (pfam00122) HAD_like, haloacid dehalogenase-like (cd04127) Arabidopsis query “Chromalveolates” Excavates HMA1: heavy metal ATPase Rhodophytes (At4g37270) Unikonts Viridiplants Prokaryotes HMA1

other C prokaryotes

87 -Methanohalophilus mahii DSM 5219 gi294495676 53 Euryarchaeota-Methanococcoides burtonii DSM 6242 gi91772641 Euryarchaeota-Methanohalobium evestigatum Z-7303 gi298674971 100 77 - lykanthroporepellens BL-DC-9 gi300088297 -Thermacetogenium phaeum DSM 12270 gi410668389 100 -planctomycete KSU-1 gi386811127 100 Euryarchaeota-Thermococcus litoralis DSM 5473 gi375082994 Euryarchaeota-Pyrococcus sp. ST04 gi389852347 Viridiplantae-Solanum lycopersicum gi460369361 51 Viridiplantae-Vitis vinifera gi225448275 Viridiplantae-Ricinus communis gi255581361 Viridiplantae-Populus trichocarpa gi224073351 Viridiplantae-Cucumis sativus gi449438779 100 100 Viridiplantae-Arabidopsis lyrata jgi857226 Viridiplantae-Arabidopsis thaliana gi42573157 PAA1 clade Viridiplantae-Fragaria vesca subsp vesca gi470108222 54 Viridiplantae-Glycine max jgiGlyma08g07710 100 Viridiplantae-Sorghum bicolor gi242082423 100 Viridiplantae-Brachypodium distachyon gi357148204 90 Viridiplantae-Physcomitrella patens subsp. patens gi168035237 mediterraneus-MMETSP0938 0179713786 EVOLUTION -Paulinella chromatophora gi194476775 Cyanobacteria-Trichodesmium erythraeum IMS101 gi113475254 68 Cyanobacteria-Arthrospira platensis NIES-39 gi479127846 98 Cyanobacteria-Lyngbya sp. PCC 8106 gi119486994 60 100 Cyanobacteria-Anabaena variabilis ATCC 29413 gi75907770 Cyanobacteria-Nostoc sp. PCC 7120 gi17231274 100 Cyanobacteria-'Nostoc azollae' 0708 gi298490874 Cyanobacteria-Nodularia spumigena CCY9414 gi119509118 Cyanobacteria-Crinalium epipsammum PCC 9333 gi428305746 99 Cyanobacteria-Crocosphaera watsonii WH 8501 gi67922678 53 98 Cyanobacteria-Cyanothece sp. ATCC 51472 gi354552434 100 Cyanobacteria-Cyanothece sp. CCY0110 gi126658952 Cyanobacteria-Moorea producens 3L gi332710730 Viridiplantae-Arabidopsis lyrata jgi490893 100 Viridiplantae-Arabidopsis thaliana gi15235511 61 Viridiplantae-Populus trichocarpa gi224094264

Viridiplantae-Ricinus communis gi255567899 HMA1 clade 93 Viridiplantae-Fragaria vesca subsp vesca gi470103122 91 Viridiplantae-Cucumis sativus gi449468396 66 62 Viridiplantae-Glycine max gi356565533 Viridiplantae-Vitis vinifera gi225438839 100 Viridiplantae-Solanum lycopersicum gi460372126 98 100 Viridiplantae-Brachypodium distachyon gi357117442 Viridiplantae-Oryza sativa Japonica Group gi115469636 98 Chlorophyta salinarum-MMETSP0807 0183833482 100 Rhodophyta- A stig 29-Gs35650 1 Rhodophyta-Chondrus crispus T00006183001 100 100 Chlamydiae- chondrophila WSU 86-1044 gi297621848 99 Chlamydiae-Parachlamydia acanthamoebae str. Hall's coccus gi282891102 Chlamydiae-Simkania negevensis Z gi338732433 76 100 Chlamydiae- abortus LLG gi424825512 Chlamydiae- psittaci M56 gi407459716 100 Chlamydiae-Chlamydophila psittaci 01DC11 gi384451969 99 Chlamydiae- NJ1 gi406593848 Chlamydiae- MopnTet14 gi301336271 - pneumophila subsp. pneumophila str Philadelphia 1 gi52841244 Firmicutes-Anoxybacillus sp. DT3-1 gi470088449 94 52 Firmicutes-Thermincola potens JR gi296131980 Firmicutes-Caldalkalibacillus thermarum TA2 A1 gi335038349 74 99 unclassifiedBacteria-Thermobaculum-Thermobaculum terrenum ATCC BAA-798 gi269926252 100 Firmicutes-Geobacillus sp. WCH70 gi239826094 99 Proteobacteria--Kingella denitrificans ATCC 33394 gi325267405 100 Firmicutes- xylanus NBRC 15112 gi408355524 Firmicutes-Geobacillus sp. Y412MC52 gi319765801 Euryarchaeota-Halogranum salarium B-1 gi399579023 Euryarchaeota-Haloarculay japonica DSM 6131 gi448688914 88100 85 Euryarchaeota 95 - sp. 7 1 gi237745313 100 -Anaerobaculum hydrogeniformans gi289522690 100 -Prevotella histicola F0411 gi357043465 Bacteroidetes-Prevotella amnii CRIS 21A-A gi307564735 Chloroflexi-Ktedonobacteria-Ktedonobacter racemifer DSM 44963 gi298241596 100 Proteobacteria-Syntrophobacter fumaroxidans MPOB gi116751190 51 100 Planctomycetes-Rhodopirellula sp. SWK7 gi470886103 Planctomycetes-Rhodopirellula sallentina SM41 gi470883805 Proteobacteria-Pseudomonas sp. GM16 gi399013333 100 Chloroflexi- DSM 9485 gi219849569 80 100 Chloroflexi- J-10-fl gi163846446 Chloroflexi-Chloroflexus sp. Y-400-fl gi222524221 Chloroflexi-Anaerolineae- thermophila UNI-1 gi320160161 Proteobacteria-Alteromonas macleodii AltDE1 gi410860838 79 100 Proteobacteria-Shewanella sp. W3-18-1 gi120597953 Deinococci- silvanus DSM 9946 gi297566049 95 Deinococci- maricopensis DSM 21211 gi320333774 100 100 Deinococci-Deinococcus gobiensis I-0 gi386854478 53 Deinococci- DSM 11300 gi94972049 Deinococci-Deinococcus peraridilitoris DSM 19664 gi429221623 Deinococci- R1 gi15807741 100 Proteobacteria- winogradskyi Nb-255 gi75677312 other prokaryotes

Proteobacteria-Gallionella capsiferriformans ES-2 gi302878165 100 100 Bacteroidetes-Spirosoma linguale DSM 74 gi284005704 100 Bacteroidetes-Echinicola vietnamensis DSM 17526 gi431799419 100 Bacteroidetes-Anaerophaga thermohalophila DSM 12881 gi346225162 96 Planctomycetes-Pirellula staleyi DSM 6068 gi283778995 Proteobacteria-Sphingomonas echinoides ATCC 14820 gi393720128 0.2 substitutions/site

Fig. 3. Network and phylogenetic analysis of the plastid targeted HMA1-PAA1 copper transporters. (A) The network that includes the ACA1/PEA1 calcium ATPase of apparent eukaryotic origin that shares a weak connection to HMA1/PAA1 transporters based on sharing (Fig. S3). The nodes in this network are labeled according to taxonomic origin within prokaryotes and eukaryotes. (B) The same network was relabeled with nodes indicating distribution in different eukaryotic phyla. This image highlights the independent prokaryotic origins of the HMA/PAA1 family in eukaryotes. (C) RaxML tree (WAG + Γ model of evolution) of the HMA1/PAA1 data with redundant sequences removed. The results of 100 bootstrap replicates are shown at the branches. Cyanobacteria are in blue text, Viridiplantae are in green text, red algae are in red text, Chlamydiae are in magenta text, and all other taxa are in black text. The query transporter sequences from Arabidopsis are shown in boldface black text within each Viridiplantae clade.

Karkar et al. PNAS | August 18, 2015 | vol. 112 | no. 33 | 10211 Downloaded by guest on October 4, 2021 Evolution of the UhpC family. A surprising finding of the analysis Here we extended this analysis by generating in silico targeting of the genome of the glaucophyte Cyanophora paradoxa is that predictions for the other Archaeplastida UhpCs to determine in contrast to red algae and Viridiplantae, it does not encode whether any of these might also be plastid destined. Our hy- NST-derived plastid-targeted sugar-phosphate transporters (Fig. pothesis was that if red and/or green algal UhpCs are also plastid S1) (6). Six nonplastidial, likely endomembrane-targeted, NST targeted, then the common ancestor of Archaeplastida was likely proteins are present in the Cyanophora genome, suggesting to have relied on this bacterium-derived protein as the primor- that the had split from the Archaeplastida before dial transporter of a C6 carbon compound that can be fed di- the gene duplications and acquisition of plastid-targeting signals rectly into cytosolic glycogen biosynthesis after being converted that propelled pPT evolution in red algae and Viridiplantae. A into UDP-glucose (i.e., the ancestral location of starch synthesis closer inspection of Cyanophora gene models, however, turned in Archaeplastida before relocation to the Viridiplantae chlo- up two novel candidates for plastid sugar-phosphate transport in roplast). This HGT event could have occurred before (or per- this species. These genes encode homologs of bacterial mem- haps coincident) with the retargeting of members of the NST brane bound UhpC-type hexose-phosphate sensors, which in family and before origin of the pPT genes (43). Use of the are part of an operon that is responsible for the uptake program ChloroP 1.1 (44) suggested that many complete UhpC of glucose-6-phosphate (G-6-P) (42). In contrast to their bacte- sequences in red and green algae are likely to be plastid targeted rial ancestors, both Cyanophora UhpC homologs feature an (Fig. 4A). We tested this hypothesis for UhpCs in the red algae N-terminal extension that displays characteristic features of Galdieria sulphuraria (45) and Cyanidioschyzon merolae (46). To glaucophyte plastid-targeting sequences (6). Analysis of the plastid this end, we expressed fluorescent-tagged fusion proteins from inner envelope of Cyanophora confirmed the presence these algae in Nicotiana benthamiana and observed their locali- of the two UhpCs in the envelope fraction (43). This finding was zation by fluorescence microscopy. The full-length proteins of further corroborated by transient expression of fluorescent- G. sulphuraria (gene Gasu_03960) and of C. merolae (gene tagged Cyanophora UhpCs in tobacco cells, where they localized to CYME_CMQ264C), as well as the first 112 and 200 amino acids the periphery of the plastids (43). Whereas NST-derived plastid of each protein corresponding to their putative transit peptides, sugar-phosphate transporters are restricted to red algae and were cloned in front of the N terminus of the yellow fluorescent Viridiplantae, it now becomes clear that UhpC is widespread in protein (YFP). Observation of the protoplast isolated from algal members of the Archaeplastida (Fig. 4A; but lost in plants). leaves infiltrated with the constructs carrying the predicted In Cyanophora, UhpC is putatively responsible for the counter transit peptides fused to the YFP shows that the fluorescent exchange of orthophosphate for G-6-P, thereby likely constituting signal colocalizes with the autofluorescence (Fig. 4B, the ancestral path of carbon export from plastids in the Archae- Plates 1 and 3). This result indicates that the red algal predicted plastida (43). transit peptides are indeed sufficient to target the YFP to the

100 reinhardtii 159482272 (+) 0.52 AB100 Chlamydomonas reinhardtii gi159482274 (+) 0.53 YFP chlorophyll merge carteri f. nagariensis gi302840937 (+) 0.57) 89 NC64A jgi141550 (-) 0.46 µ 100 Coccomyxa subellipsoida jgi52341 (+) 0.55 20 m

Chlorella vulgaris jgi81415 (+) 0.55) Archaeplastida 100 Asterochloris sp. jgi24630 (-) 0.44 sp. RCC299 gi255070251 (+) 0.55 Micromonas pusilla CCMP1545 gi303273388 (+) 0.51 92 100 Ostreococcus RCC809 jgi90209 (-) 0.46 gi308800194 (+) 0.52) 99 Ostreococcus lucimarinus CCE9901 gi145343717 (+) 0.56 100 100 Cyanophora paradoxa contig37408 (incomplete) Cyanophora paradoxa contig54038 (+) 0.55 1 52 Gloeochaete wittrockiana-MMETSP1089 0184335576 (+) 0.55 Galdieria sulphuraria stig 24-Gs31310.1 (+) 0.51 20µm 100 Cyanidioschyzon merolae CMQ264C (+) 0.52 Boldia erythrosiphon contig 6072 2 (incomplete) 86 Hildenbrandia pulchella contig 40013 4 (+) 0.57 53 87 87 Calliarthron tuberculosum ORF 2866 133 (incomplete) 71 Chondrus crispus T00007675001 (-) 0.49 100 Porphyridium purpureum contig2054.9 (-) 0.44 Dixoniella grisea contig 7817 3 (incomplete) 100 Candidatus Protochlamydia amoebophila UWE25 gi46446021 92 Parachlamydia acanthamoebae UV-7 gi338176229 2 Waddlia chondrophila WSU 86-1044 gi297621742 Chlamydophila pneumoniae AR39 gi16752375 20µm 100 Chlamydophila felis Fe/C-56 gi89898737 Chlamydophila caviae GPIC gi29839842 80 Chlamydophila abortus S26/3 gi62184720 69 Chlamydophila psittaci Cal10 gi329942393 Chlamydia psittaci CP3 gi406591890 Chlamydophila psittaci 01DC11 gi384451181 Chlamydia psittaci 01DC12 gi410858063 Chlamydia psittaci NJ1 gi406593001 GammaLegionella pneumophila str. Lens gi54295389 3 2300/99 Alcoy gi296108199 µ 100 Legionella pneumophila str. Corby gi148359980 20 m Fluoribacter dumoffii Tex-KL gi388455407 88 D-4968 gi270157197 Legionella drancourtii LLAP12 gi374261936 Aggregatibacter actinomycetemcomitans D11S 1 261866887 100 82 Other Proteobacteria 0.5 substitutions/site 4

Fig. 4. Analysis of UhpC proteins in Archaeplastida. (A) RaxML tree (WAG + Γ model of evolution) with the results of 100 bootstrap replicates shown at the branches. Glaucophyta are in blue text, Viridiplantae are in green text, red algae are in red text, Chlamydiae are in magenta text, and all other prokaryotes are in black text. The four proteins for which localization data exist to indicate plastid inner envelope membrane targeting are shown in large boldface black text. The results of bioinformatic targeting predictions using ChloroP (cTP score; marked with [+] when predicted to be plastid targeted or [−] when not) are shown for each Achaeplastida UhpC, when complete proteins are available. (B) Expression of YFP-fusion constructs in Nicotiana benthamiana protoplasts. Confocal microscope pictures on isolated Nicotiana protoplasts expressing the YFP-fusion constructs driven by the ubiquitin 10 promoter. (Plate 1) Expression pattern of the predicted transit peptide of the UhpC homolog Gasu_03960 from Galdieria fused to YFP. Shown are YFP fluorescence (YFP) in green, chlo- rophyll autofluorescence (chlorophyll) in red and an overlay of the two pictures (merge). (Plate 2) Expression pattern of the full-length Gasu_03960 fused to YFP. (Plate 3) Expression pattern of the predicted transit peptide of the UhpC homolog CYME_CMQ264C from Cyanidioschyzon fused to YFP. (Plate 4)Ex- pression pattern of the full-length CYME_CMQ264C fused to YFP.

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chloroplast. The fluorescent signal of the full-length proteins C2-pathway (48, 49). The capacity to detoxify 2-PG inside the COLLOQUIUM fused to the YFP for Gasu_03960 and CYME_CMQ264C sur- plastid must have been lost early during endosymbiosis because rounds the plastids, a pattern typical for proteins located to the all members of the Archaeplastida rely on the export of glycolic inner envelope membrane (Fig. 4B, Plates 2 and 4). This obser- acid from plastids to the cytoplasm for conversion to glycerate in vation is consistent with localization to the plastid inner envelope mitochondria and/or . This process requires the export membrane as previously described (43). of glycolic acid from, and the import of glycerate into, the plastid, These results can also interpreted from the perspective of the which is achieved by the recently discovered glycolate/glycerate ménage à trois hypothesis of symbiont integration (37, 47). This transporter PLGG1, a eukaryote-derived protein (Table S1). hypothesis posits that plastid endosymbiosis putatively relied on Some cyanobacteria are able to excrete glycolate at significant three partners: the host, the cyanobacterium, and a chlamydial levels, with 9% of net fixed carbon lost in air for heterocystous symbiont (hence, ménage à trois). Under this view, the UhpC species and up to 60% lost in some strains under high oxygen transporter (of bacterial origin; see below) could have been conditions (50). This mechanism, however, represents a significant transferred first to the cyanobacterium genome to allow the flow loss of carbon and of Calvin–Benson cycle intermediate products. of fixed carbon to the inclusion vesicle (of chlamydial origin) that Hence, recovery of part of this carbon by the eukaryotic photo- housed both prokaryotes. The G-6-P that was secreted from respiration pathway would offer a significant selective advantage. the cyanobacterium, after conversion to G-1-P by phosphoglu- The advantage of PLGG1 over other (unknown) cyanobacterial comutase, acted as substrate for the synthesis of either ADP- or glycolate exporters is that it works in a counter exchange mode, UDP-glucose and subsequently glycogen in the vesicular space. which leads to the salvage of three of four carbon units lost Glycogen was also synthesized in the host cytosol with the use of via oxygenation/photorespiration. chlamydial-derived (GlgA, GlgC) secreted by their type In summary, the metabolic connectivity model we present here 3 secretion system (TTS). Regardless of the manner in which the relies on functional diversification of organelle functions. ancient ménage à trois interaction operated, over time the UhpC Through targeting of host-derived transporter proteins to the (and other endosymbiont) genes were transferred to the host organelle membrane, two previously distinct metabolic networks genome and retargeted to the plastid inner membrane to allow become increasingly interwoven and interdependent. This idea is uptake of G-6-P for glycogen synthesis. Therefore, genes pro- supported by previous work that shows the growth of metabolic vided to the host by chlamydial cells, other bacteria, and the networks in prokaryotes is frequently achieved by the addition of cyanobacterium forged the successful endosymbiosis, and the transporter activities at the network periphery. That is, given a EVOLUTION chlamydial symbiont (and their inclusion vesicles) was lost (47). core set of metabolic functions, the network can be expanded The phylogenetic evidence for a chlamydial origin of UhpC in by gaining access to additional substrates via the acquisition of Archaeplastida is ambiguous because of the presence of many genes encoding transporter proteins. These elaborations are Proteobacteria in the tree that also forms a sister to the eukaryote driven by to changing environments (e.g., extracel- clade. Regardless of the rooting scheme that is used, the direction lular vs. intracellular) (51). In addition, it is widely recognized of HGT in this case is, however, clear. Among eukaryotes, only that free-living bacteria have access to a large pan-domain gene Archaeplastida contain a bacterium-derived UhpC gene; therefore, pool from which they can acquire novel metabolic and trans- the transfer was most likely from a to a eukaryote cell. porter genes. Therefore, the cyanobacterial plastid ancestor had a chimeric genome at the time of endosymbiosis, compo- The Metabolic Connectivity Model. Given the strong evidence for nents of which were transferred to the eukaryote host by EGT. eukaryotic or HGT-derived metabolite transporters in Archae- The vast prokaryotic gene pool also contributed to Archaeplastdia plastida, both of which are nuclear encoded, how might these evolution via independent HGT events. The role of HGT in en- proteins be directly implicated in early events in endosymbiont dosymbiont integration is of particular relevance because once integration? For clues to answering this question, we can look at cells are internalized they rarely gain genes, but rather undergo the recent plastid endosymbiosis in Paulinella. Work done by significant genome reduction (12–14). Hence, it is not surprising Nowack and Grossman (26) has shown that this appar- that, as we show here, growth in functional diversity of plastids ently relies on the secretory system, via passage through the Golgi, is achieved by the retargeting of host encoded proteins or the to deliver essential proteins (PsaE, PsaK1, PsaK2) to acquisition by the host of foreign genes whose products can be the chromatophore. These proteins have been lost from the en- retargeted to the organelle. dosymbiont genome and are now encoded in the nucleus, synthe- sized in the cytoplasm, and trafficked into the chromatophore. The Methods absence of a detectable terminal or internal targeting signal in Network and Phylogenetic Analysis. The 34 different validated A. thaliana these proteins suggests that the secretory system is capable of de- plastid inner membrane transporter sequences listed in Fischer (25) and the livering mature proteins to an endosymbiont. We take these results three new transporters (27, 28) were used in our analyses. Each protein se- as support for the idea that the initial metabolite transporters that quence from Arabidopsis was used to query via BLASTP an in-house data- serviced the Archaeplastida (as described above) plastid could also base composed of The National Center for Biotechnology Information have been delivered by the secretory system to the inner envelope Reference sequence database (RefSeq) v59, the 672 publicly available from the Moore Marine Eukaryote membrane to allow exchange of metabolites with the host cell (20). Project (MMETSP; camera.calit2.net/mmetsp/list.php), with additional eu- Our work shows that the UhpC hexose-phosphate transporter karyote proteomes obtained from NCBI dbEST, (www.ncbi.nlm.nih.gov/ present in the Archaeplastida ancestor could have been an early dbEST/), and TBestDB. The BLASTP output for each transporter protein se- player in fixed carbon transport, as envisioned in the ménage à quence was analyzed so that a maximum of 100 individual hits was retained trois hypothesis, or under a scenario that does not rely on an in- with a limit of ≤12 hits per to avoid oversampling taxon-rich phyla in clusion vesicle as the initial platform for glycogen synthesis. the database. Each dataset was aligned using Clustal Omega (52), and The hypothesis of a host-dominated process for metabolite phylogenetic trees were constructed with the full alignment using IQ-TREE transport in the nascent plastid endosymbiont as postulated here v.0.9.6 (53) with automatic best-fit model selection and 1500 μLtra-fast would be greatly strengthened by evidence that crucial functions bootstrap replicates (Fig. S2). We also used Gblocks (54) to trim each trans- porter alignment to conserved sites using a block size of 2 (−b4 = 2) and were provided by the eukaryote. Another example of such a allowing all gaps (−b5 = a). These reduced alignments were used as input to function is the essential detoxification of the oxygenation prod- IQ-TREE as described above to generate phylogenies and bootstrap support uct of , 2-phosphoglycolic acid (2-PG). Cyanobacteria values. For some transporters (e.g., pPTs, PTs), the individual protein align- possess at least three distinct metabolic routes to this end, ments were combined, and a RAxML (55) tree was inferred using the WAG + Γ including the bacterial-type glycolate pathway and a plant-like model of sequence evolution and 100 bootstrap replicates.

Karkar et al. PNAS | August 18, 2015 | vol. 112 | no. 33 | 10213 Downloaded by guest on October 4, 2021 For the network analysis, the complete set of transporters that was clustering allows us to detect sequences that are related through intermediates returned from the phylogenomic analysis was analyzed to remove all re- that would not be present in the same tree due to high pairwise sequence dundant sequences. This data set of 2,330 unique sequences was used in an all divergence between the most distantly related protein families. This property − vs. all BLASTP analysis with a minimum e-value cutoff ≤10 5; i.e., a threshold potentially allows us to merge regions of different trees into single clusters to that selected hits based on both bit score (the similarity measure) and identify ancient connections between transporters. We have previously applied length. This restriction returned short hits to regions with high similarity this principle to detect ancient signals of monophyly between redox enzymes, while allowing hits to longer regions with lower similarity. The lowest sim- allowing inferences into their origin and evolution in divergent prokaryotic ilarity value recorded for the hits was 18.15% identity. From these results, lineages (58–60). Here the networks were used in a more limited fashion to we applied a cutoff of 70% query coverage (i.e., any hit must cover at least potentially identify transporter family relationships that may not be revealed 70% of the 2 proteins), resulting in a set of 21 connected components that using phylogenetic methods. encompass 1,727 proteins. The classification of proteins in these connected components was done using a greedy clustering algorithm based on the Localization of Transporter Proteins in Transiently Transformed Tobacco Cells. modularity (ratio of intra- vs. intergroup links) (56) that detects densely The coding sequences of the UhpC homologs from Galdieria sulphuraria connected regions of sequences. New classes of transporters were manually (Gasu_03960) and Cyanidioschyzon merolae (CYME_CMQ264C) were ampli- defined to regroup transporter families listed in Fischer (25) that have ho- fied by PCR and cloned by Gibson cloning into the plant expression vector mologs that belong the same community in the network. The taxonomic pUBC-YFP for C-terminal YFP-fusion under the control of the ubiquitin content of the network was analyzed and displayed using in-house R scripts 10 promoter (61, 62). Agrobacterium tumefaciens transformation (strain (57) with the igraph package (Figs. 2 and 3). GV3101), tobacco leaf infiltration, and protoplast isolation were done as The motivation to use both phylogenetic and network methods with the previously described (43). Isolated protoplasts were observed 2–4dafter same data reflects the fact that these approaches provide complementary in- infiltration with an inverted Zeiss LSM 780 confocal laser-scanning mi- sights into transporter evolution. Phylogenetic methods assume that sequences croscope. YFP and chlorophyll were excited using the 488-nm laser line of are inherited via vertical descent and the goal here was to detect these instances, an Argon laser, and the emission was collected at 517–578 and 590–690 nm, as well as subsets of the data that contradict this assumption due to well-resolved respectively. cases of endosymbiotic or horizontal gene transfer. In contrast, network analysis is a straightforward clustering approach with input sequences, in which no ACKNOWLEDGMENTS. This research was funded by National Science assumption is made regarding the mechanism of evolution. Sequence clustering Foundation Grants 0936884 and 1317114 (to D.B.). A.P.M.W. appreciates is based on several criteria (e.g., hit coverage, similarity score) using a robust support from the Deutsche Forschungsgemeinschaft (Grants EXC 1028 and algorithm that detects densely connected sets of proteins. Importantly, the WE 2231/8-2).

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