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REVIEW ARTICLE published: 17 October 2014 ECOLOGY AND EVOLUTION doi: 10.3389/fevo.2014.00066 Primary endosymbiosis and the evolution of light and oxygen sensing in photosynthetic

Nathan C. Rockwell 1, J. C. Lagarias 1 and Debashish Bhattacharya 2*

1 Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA, USA 2 Department of Ecology, Evolution, and Natural Resources; Institute of Marine and Coastal Science, Rutgers University, New Brunswick, NJ, USA

Edited by: The origin of the photosynthetic organelle in eukaryotes, the , changed forever the Bernd Schierwater, TiHo Hannover, evolutionary trajectory of on our planet. are highly specialized compartments Germany derived from a putative single cyanobacterial primary endosymbiosis that occurred in Reviewed by: the common ancestor of the supergroup that comprises the Denis Baurain, Université de Liège, Belgium (green and ), , and algae. These lineages include James Cotton, Wellcome Trust critical primary producers of freshwater and terrestrial ecosystems, progenitors of which Sanger Institute, UK provided plastids through secondary endosymbiosis to other algae such as diatoms and Dion G. Durnford, University of New dinoflagellates that are critical to marine ecosystems. Despite its broad importance and Brunswick, Canada the success of algal and lineages, the phagotrophic origin of the plastid imposed *Correspondence: Debashish Bhattacharya, an interesting challenge on the predatory eukaryotic ancestor of the Archaeplastida. By Department of Ecology, Evolution engulfing an oxygenic photosynthetic , the host lineage imposed an oxidative stress and Natural Resources and Institute upon itself in the presence of light. Adaptations to meet this challenge were thus likely to of Marine and Coastal Science, have occurred early on during the transition from a predatory phagotroph to an obligate Rutgers University, 59 Dudley Road, Foran Hall 102, New Brunswick, (or ). Modern algae have recently been shown to employ linear NJ 08901, USA tetrapyrroles (bilins) to respond to oxidative stress under high light. Here we explore the e-mail: debash.bhattacharya@ early events in plastid evolution and the possible ancient roles of bilins in responding to gmail.com light and oxygen.

Keywords: algal evolution, Archaeplastida, phytochrome, ferredoxin-depending bilin reductase, phagotrophy, primary endosymbiosis

IT’S COMPLICATED: THE STORY OF PLASTID PRIMARY multi-gene data have suffered from a litany of woes including ENDOSYMBIOSIS highly diverged genes that retain poor phylogenetic signal, long All multicellular life on our planet ultimately relies on organisms branch attraction artifacts, and horizontal or endosymbiotic gene that have the ability to convert solar energy into carbohydrates transfer (HGT, EGT) that can generate a reticulate evolutionary through oxygenic (Cavalier-Smith, 1982; Palmer, history for genes. This situation is made even more compli- 2002; Bhattacharya et al., 2004; Falkowski et al., 2004), despite the cated by an unknown history of gene duplication and loss that formation of reactive oxygen (ROS) as toxic byproducts sometimes makes the identification of gene orthologs difficult (Niyogi and Truong, 2013; Goss and Lepetit, 2014). Figuring out (Hackett et al., 2007; Stiller, 2007). Although important for cell how and when the powerhouse of photosynthesis, the plastid, first evolution and adaptation, these processes confound an unbiased entered the eukaryotic via primary plastid endosymbio- assessment of Archaeplastida monophyly (e.g., Burki et al., 2007; sis has proven challenging. Three major groups contain what are Patron et al., 2007; Parfrey et al., 2010; Grant and Katz, 2014). termed “primary” plastids surrounded by a double membrane: Alternative approaches such as studying multi-protein complexes , rhodophytes (red algae), and the Viridiplantae or cataloging the origins of individual genes in genome-wide ( and land plants). These lineages are putatively united gene inventories are therefore increasingly prevalent in studies in the monophyletic supergroup Archaeplastida (Adl et al., 2012 of the evolutionary history of primary-plastid-containing algae [also known as Plantae]), with their common ancestor having (e.g., Chan et al., 2011; Price et al., 2012). captured the plastid via phagotrophic engulfment of a free- These studies have addressed the fundamental issue of a single living cyanobacterium (Figure 1). Although initially supported or up to three independent primary endosymbiotic events giv- by plastid gene and genome phylogenies, the monophyly of ing rise to the Archaeplastida plastid. This “numbers game” with Archaeplastida is not conclusively demonstrated by nuclear gene primary endosymbioses seems trivial as such but has far deeper data or host cell ultrastructure (Rodríguez-Ezpeleta et al., 2005; consequences when seen from the perspective of organellogene- Hackett et al., 2007; Kim et al., 2014). Both of these types of sis. This is explained by the fact that plastids are not autonomous inferences are weakened by the >1 billion years of evolution that entities but rather, profoundly integrated into and dependent have passed since the divergence of Archaeplastida lineages (e.g., on host cell biology. Plastids have highly reduced genomes Yoon et al., 2014). Specifically, phylogenetic made using (ca. 100–200 Kbp in size, compared to ≥ 1.6 Mb for free-living,

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translated proteins from the into the plastid to carry out their functions (Jarvis and Soll, 2002; Reumann et al., 2005; Gross and Bhattacharya, 2008, 2009). Phylogenetic analysis of shared plastid translocons, comprising at least 12 different proteins in Archaeplastida (Shi and Theg, 2013), provides strong evidence for a monophyletic origin of the nanomachines that control plas- tid protein import. When viewed from this perspective, we can ask the question: what are the chances that the genes encoding such plastid-localized, multi-protein complexes could have origi- nated multiple times in the constituent lineages? Under the most parsimonious view, the genes were assembled in a single com- mon ancestor, and therefore Archaeplastida are descended from such a single ancestor (i.e., are monophyletic: McFadden and van Dooren, 2004; Kalanon and McFadden, 2008; Price et al., 2012). Another landmark trait linked to plastid establishment is the coordination of carbon metabolism between the host and plastid, a process that relies on -phosphate transporters. Previous work showed that plastid-targeted sugar transporters of rhodophytes and Viridiplantae evolved from existing host endomembrane nucleotide sugar transporters (NSTs) through gene duplication, divergence, and retargeting to the photosyn- thetic organelle (Weber et al., 2006; Colleoni et al., 2010). Surprisingly, although six endomembrane-type NST genes were present in the nuclear genome of the glaucophyte , there were no plastid-targeted phosphate-translocator (PT) genes (Price et al., 2012). The search for the missing genes turned up two candidates that encode homologs of bac- terial UhpC-type hexose-phosphate transporters that were also found in other Archaeplastida. Both C. paradoxa UhpC homologs FIGURE 1 | Endosymbiotic origin of the Archaeplastida plastid through encoded an N-terminal extension that could serve as a plastid cyanobacterial primary endosymbiosis. (top) A heterotrophic engulfed free-living for food (). Over time, this targeting sequence. Surprisingly, both of the UhpC genes were situation changed, with the cyanobacterium becoming an apparently derived via HGT in the Archaeplastida ancestor from (bottom). A chlamydial cell is believed to have also been resident in the lineages related to or (Figure 2). The host at the time of endosymbiosis and provided functions critical to plastid alternative scenario of a HGT origin of the UhpC genes in, integration (Ball et al., 2013). Both of these prokaryotes gave rise to nuclear for example, Chlamydiae from an Archaeplastida source is the- genes in the Archaeplastida host through endosymbiotic gene transfer (EGT; cyanobacterium) and (HGT; chlamydial cell oretically possible. However, the type of long-term association and other ). After their split, the red and green algae gave rise to the posited for chlamydiales parasites and the Archaeplastida ances- plastid in other algae through independent secondary endosymbiosis tor has been reported in other extant (e.g., Acanthamoeba (Bhattacharya et al., 2004; Curtis et al., 2012). The intracellular transfer of castellani, Schmitz-Esser et al., 2008) and is supported by the genes via EGT and HGT is indicated (arrows). Genetic material of foreign finding of several dozen HGTs that are shared between these origin in the nucleus is shown as stripes of different colors with the color indicating the source of the gene. lineages (Qiu et al., 2013a). The easiest explanation for this sur- prising result is that genes moved in the direction: intracellular prokaryotic endosymbiont to host (see also below). photosynthetic cyanobacteria), with many genes either lost out- It thus is currently hypothesized that bacterially derived trans- right or moved to the host nucleus through EGT (Martin and port proteins provided an early means for integrating host and Herrmann, 1998; Stegemann et al., 2003; Timmis et al., 2004; plastid metabolism that persists in glaucophytes but that may Reyes-Prieto et al., 2008). These organelles rely on the host to been largely or completely replaced in other lineages of the provide energy, supply metabolites to sustain plastid functions Archaeplastida. (e.g., Weber et al., 2006), and synthesize ca. 90% of the pro- Analysis of the plastid proteome from C. paradoxa provided teins that support plastid metabolism, including Calvin cycle further support for the absence of typical NST-derived sugar proteins (e.g., Reyes-Prieto and Bhattacharya, 2007). The total transporters and the presence of the prokaryotic UhpC trans- inventory can vary from ca. 800 plastid proteins in some algae porters in the inner membrane of the plastid of this species (Facchinelli et al., 2013; Qiu et al., 2013a)to>2000 in plants (e.g., (Facchinelli et al., 2013). A similar story of chlamydial gene repur- Martin et al., 2002; see http://ppdb.tc.cornell.edu/). Many multi- posing to generate a key Archaeplastida function is provided by protein complexes are encoded entirely in the nucleus including the origin of synthesis (Huang and Gogarten, 2007; Becker the Translocons at the Inner and Outer (plastid) et al., 2008; Moustafa et al., 2008). In the transition from glyco- membranes (the TIC and TOC complexes) that shepherd newly gen to starch storage in the Archaeplastida ancestor, there is now

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FIGURE 2 | A chlamydial or proteobacterial origin for plastid details, see Price et al., 2012). Bootstrap values (when ≥ 50%) are shown at hexose-phosphate transporters. Maximum likelihood (PhyML) phylogeny of the branches. Red algae, Viridiplantae, glaucophytes, and Chlamydiae are UhpC-type hexose-phosphate transporters in algae, plants, and bacteria (for shown in red, green, magenta, and blue text, respectively. evidence that genes encoding key effector molecules such as GlgX plastid endosymbiosis. How did the heterotrophic ancestor of debranching enzyme were provided by a chlamydial partner (Ball the Archaeplastida sense and deal with the toxic effects of oxy- et al., 2013). The importance of host, cyanobacterial, and chlamy- gen during a time when cyanobacteria presumably dominated dial components in forging the plastid (the “ménage à trois” aquatic ecosystems? Specifically, the phagotrophic origin of the hypothesis of Ball et al., 2013) has been proposed as an expla- primary plastid necessitated intimate contact between the host nation of the rarity of photosynthetic primary endosymbiosis in and oxygen-evolving, light capturing prey. Furthermore, once eukaryotes (the only other case being the plastid-containing filose prey toxicity was dealt with, how did oxygen and light sens- chromatophora: Nowack et al., 2008; Yoon ing evolve to become integrated to support a highly coordi- et al., 2014). nated photosynthetic lifestyle that is widespread in the world’s In summary, current understanding strongly supports (but oceans? Recent findings begin to answer these fundamental does not prove) a single primary endosymbiosis in Archaeplastida questions and will form the focus of the remainder of this followed by complex, perhaps reticulate genome evolution. HGT perspective. from bacterial sources and EGT from the endosymbiont played major roles in shaping gene content and plastid function (e.g., HOW DID THE ARCHAEPLASTIDA ANCESTOR SENSE LIGHT Chan et al., 2012; Price et al., 2012; Ball et al., 2013; Qiu et al., AND OXYGEN? 2013a,b,c;seeFigure 1). The putative role of chlamydial or other Oxygenic photosynthesis is a vital reaction on our planet. prokaryotic parasites in facilitating plastid integration provides However, this boon comes at a cost to algal and plant cells. another layer of complexity to the story. A key piece of the Light harvesting can capture excess energy that must be dis- puzzle that remains unresolved involves the earliest events in sipated, and oxygen evolution during photosynthesis supports

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formation of ROS that need to be detoxified (Halliwell, 2006; stress-response genes requires the existence of retrograde signal- Knoefler et al., 2012). The importance of such detoxification ing pathways to report the state of the plastid to the nucleus for is clearly shown by the evolution of a range of mechanisms regulation of stress-response genes. for non-photochemical quenching (NPQ) in modern photosyn- Light-harvesting use linear tetrapyrroles thetic organisms (Niyogi and Truong, 2013; Goss and Lepetit, (bilins) as chromophores. Reduced phytobilins and 2014). Photosynthetic organisms must balance light harvesting (hereafter, bilins) are synthesized from heme via the action and ROS detoxification challenges under transient, diurnal, and of a heme oxygenase (HO) and a ferredoxin-dependent bilin seasonal changes in light intensity and temperature, in the pres- reductase (FDBR: Frankenberg et al., 2001; Dammeyer and ence of competition for sunlight from neighboring organisms, Frankenberg-Dinkel, 2008), with different FDBRs producing and during ongoing and herbivory (Rockwell et al., different bilins (Figure 3). Intriguingly, phycobiliproteins and 2006; Franklin and Quail, 2010; Casal, 2013). Moreover, there FDBRs were both cyanobacterial innovations, and bilins can is growing evidence that many algal lineages retain the capacity be present in considerable excess in cyanobacterial cells relative for mixotrophy, including ongoing ingestion of oxygenic pho- to . Bilins have known roles both in light harvest- tosynthetic prey species. Prasinophyte, cryptophyte, , ing and as photoreceptor chromophores in cyanobacterial cells and algae are all known to engulf bacteria, (Glazer, 1988; Rockwell and Lagarias, 2010). The FDBR PcyA is whereas and dinoflagellate algae are capable of engulf- apparently essential for cyanobacteria, in contrast to core phy- ing both bacteria and eukaryotic algae (Stoecker et al., 1997; cobiliproteins (Alvey et al., 2011). This surprising requirement Roberts and Laybourn-Parry, 1999; Moestrup and Sengco, 2001; for bilins, independent of light harvesting, contrasts with the Adolf et al., 2006; Burkholder et al., 2008; Van Donk et al., 2009; existence of cyanobacterial lineages that lack known bilin-based Jeong, 2011; Jeong et al., 2012; Maruyama and Kim, 2013; Gast photoreceptors. Both glaucophyte and rhodophyte algae have et al., 2014; McKie-Krisberg and Sanders, 2014; Unrein et al., retained bilin-based light harvesting, as has P. chromatophora 2014). Such mixotrophic algae must balance phototrophic and (Yoon et al., 2014). Viridiplantae have retained bilin biosynthesis phagotrophic metabolism in response to complex photobiolog- in the absence of phycobiliproteins and even in multiple lin- ical and environmental cues. Mixotrophic organisms also face eages lacking phytochromes (Figure 4), a situation reminiscent of transient oxidative stresses specific to ingestion of photosynthetic the requirement for bilin biosynthesis in cyanobacteria. In one prey in light, due to the release of photodynamic member of the Viridiplantae, the unicellular chlorophyte alga upon prey digestion. Thus, the ability to sense and respond to reinhardtii, bilins are implicated in responding changes in light quantity and quality is a fundamental driving to oxidative stress under high light and in light-dependent accu- force in algal and and is closely linked to oxidative mulation of photosynthetic antennae complexes (Duanmu et al., stress responses. In the broadest sense, light provides the energy to 2013). Moreover, recent high-throughput transcriptomic stud- power modern ecosystems but places strong selective constraints ies of eukaryotic algae (Keeling et al., 2014) have demonstrated on genome evolution in photosynthetic branches of the the apparent presence of FDBRs, and hence of bilin biosynthe- of life. sis, in all known lineages of secondarily photosynthetic algae for At the time of the original primary endosymbiosis in which ≥ 3 datasets are available (Figure 4). Thus, the extant roles Archaeplastida, the only known oxygen-producing organisms and distribution of bilins in photosynthetic eukaryotes are consis- were cyanobacteria. Most modern cyanobacteria use phyco- tent with ancient and modern roles in sensing light and oxygen. biliprotein antennae for light harvesting, as do the glaucophyte For the phagotrophic predator that was the ancestor of modern and rhodophyte lineages of the Archaeplastida. However, many Archaeplastida, before stable endosymbiosis, bilins would only cyanobacteria substitute chlorophyll-based systems for phyco- be present after ingestion of cyanobacterial prey and thus could biliproteins under iron starvation, and some cyanobacteria only have provided a biomarker for such prey. Below we explore the possess such systems (Bibby et al., 2001; Boekema et al., 2001). hypothesis that the ancestral predator exploited bilins for light Thepresenceofchlorophyllb in “green cyanobacteria” and in and oxygen sensing. plants has led to the proposal that both light-harvesting systems were present in the cyanobacterium that gave rise to the modern BILIN BIOSYNTHESIS: EVERYWHERE A BILIN, BUT plastid, with subsequent loss of Chl b synthesis in cyanobacteria APPARENTLY NOT THE SAME ONE that retain the (Pinevich et al., 2012). Ingesting Tetrapyrrole biosynthesis proceeds via a conserved trunk pathway cyanobacterial prey must have exposed predatory eukaryotes to that leads to protoporphyrin IX, the last common precursor of transient changes in oxygen tension, and subsequent digestion of chlorophyll and heme (Figure 3). Incorporation of iron produces the cyanobacterial cell would release the tetrapyrrole pigments heme, whereas incorporation of magnesium instead produces the from the photosynthetic reaction centers and light-harvesting first committed precursor for chlorophyll biosynthesis. The first complexes. These challenges would become more pronounced as step in heme breakdown is the oxidative ring opening of heme the Archaeplastida ancestor maintained living, photosynthetically to produce free iron, carbon monoxide, and biliverdin IXα (BV, active and hence became exposed to prolonged Figure 3). This reaction is carried out by HO, and HO enzymes oxidative stress in the presence of light, necessitating the rise of producing the α isomer of BV are subject to rate-limiting product detoxification mechanisms. Oxidative stress is greatest in the plas- release and hence to product inhibition (Wegele et al., 2004). In tid, but most stress-response genes are encoded in the nucleus. mammals, product inhibition is relieved by the subsequent con- Therefore, deployment of photoprotective mechanisms such as version of BV into bilirubin by biliverdin reductase, a reaction also

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FIGURE 3 | Biosynthesis of bilins. A common tetrapyrrole pathway the same reaction (reduction) on different parts of the tetrapyrrole gives rise to both heme and chlorophyll (top). Breakdown of heme (bottom). This difference in regiospecificities allows production of a proceeds via action of a heme oxygenase (HO) and a range of bilins with different spectral properties from a single ferredoxin-dependent bilin reductase (FDBR). Different FDBRs carry out precursor. known in cyanobacteria and possible in certain plants (Schluchter PcyA, HY2, PebS, and PebB all can reduce the C3 endo-vinyl and Glazer, 1997; Pirone et al., 2009, 2010). Oxygenic photosyn- side-chain to an ethylidene (Figure 3). Similarly, PebA, PebS, and thetic organisms also have BV turnover through the action of PUBS all can reduce the 15,16-double bond separating the C- FDBRs (Figure 3). and D-rings of the linear tetrapyrrole. However, there are also To date, six different FDBRs have been identified: PcyA, HY2, unique properties that distinguish the different FDBRs. For exam- PebA, PebB, PebS, and PUBS (Dammeyer and Frankenberg- ple, only PcyA can reduce the C18 exo-vinyl side-chain of BV, Dinkel, 2008; Chen et al., 2012). PebS has only been reported and only PUBS can reduce the 4,5-double bond separating the in phages (Dammeyer et al., 2008). The six FDBRs all carry A- and B-rings (Figure 3). Whereas HY2 and PebB carry out out reduction of linear tetrapyrroles, but they reduce different the same reaction, they exhibit distinct substrate specificity: HY2 parts of the substrate; that is, different FDBRs exhibit different converts BV into phytochromobilin (PB) and cannot act on regiospecificities to produce a range of bilins (Figure 3). PcyA, 15,16-dihydrobiliverdin (15,16-DHBV), whereas PebB converts PebS, and PUBS all carry out four-electron reductions, whereas 15,16-DHBV into phycoerythrobilin (PEB) and cannot act on BV HY2, PebA, and PebB instead carry out two-electron reductions. (Frankenberg et al., 2001; Kohchi et al., 2001; Dammeyer and There is some overlap in the regiospecificities of these enzymes. Frankenberg-Dinkel, 2006).

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FIGURE 4 | Distribution of phytochromes and FDBRs in eukaryotic BLAST searches of genomic and transcriptomic data (with default algae. Evolution of primary algae (Archaeplastidae) is shown, with parameters) using the cyanobacterial phytochrome Cph1 as a query glaucophyte (blue), rhodophyte (red) and Viridiplantae (green) lineages. We sequence. The presence of FDBRs was assessed using a similar strategy, define subsequent evolution of the Viridiplantae with an initial split into with PcyA from Anabaena sp. strain PCC 7120 and PebA and PebB from streptophytes and prasinophytes. The streptophytes comprise modern Nostoc punctiforme as query sequences. FDBRs were assigned to the charophytes and land plants (). Modern charophyte and PcyA, PebA, or PebB lineage based on BLAST scores with the three query prasinophyte algae are paraphyletic, with land plants and chlorophyte algae sequences, an approach that provided both unambiguous assignment of descending from charophytes and prasinophytes, respectively (Worden algal FDBRs and recovery of the three lineages detected previously (Chen et al., 2009; Timme et al., 2012; Duanmu et al., 2014). Subsequent et al., 2012). The presence of PcyA only in certain divisions within endosymbioses (dashed gray arrows) gave rise to secondary algae rhodophytes and cryptophytes is indicated by the asterisk (e.g., presence (parentheses), which are color-coded to indicate the Archaeplastida lineage of PcyA in the cryptophyte ). Question marks indicate that was assimilated. Tertiary endosymbioses of diatoms by dinoflagellates groups for which complete draft genomes and/or >3 transcriptomic are not shown. Distribution of phytochromes was assessed by performing datasets are not yet available.

Modern cyanobacteria contain PcyA, PebA, and PebB. The 2012; Hori et al., 2014). At some point during evolution of presence of all three enzymes in different Archaeplastida lin- Viridiplantae, PebA and PebB underwent changes and became the eages (Figure 4) is consistent with their existence in the plastid modern enzymes PUBS and HY2, respectively (Figure 4). In both ancestor. Glaucophytes retain PcyA, consistent with their use streptophytes and prasinophyte/chlorophyte algae, modern PebA of its (PCB) product (Figure 3) both in light- relatives acquired the ability to reduce the 4,5-double bond of BV harvesting proteins and in phytochrome photosensors (Lemaux in addition to the 15,16-double bond, resulting in a transition and Grossman, 1985; Rockwell et al., 2014). Whereas some red from PebA to PUBS. PUBS was subsequently lost in flowering algal extremophiles such as merolae also possess plants but retained in prasinophytes (Chen et al., 2012). It thus PcyA (Nozaki et al., 2007), most rhodophytes retain only PebA seems likely that the transition from PebA activity to PUBS activ- and PebB to provide chromophores for their light-harvesting ity occurred after the divergence of rhodophytes but before the phycobiliproteins (Bhattacharya et al., 2013; Collén et al., 2013; split between streptophyte and prasinophyte algae. Prasinophytes Nakamura et al., 2013; Schönknecht et al., 2013). Within the initially retained both PUBS and PcyA, whereas PcyA is the only Viridiplantae, streptophytes initially retained representatives of FDBRfoundinmodernchlorophytes(Chen et al., 2012; Duanmu the PebA and PebB lineages; a conclusion confirmed by analyses et al., 2013). Transcriptomic studies suggest that reduced pico- of charophyte and the genome of the filamen- prasinophyte genomes such as in and tous charophyte alga Klebsormidium flaccidum (Timme et al., sometimes retain only PUBS (Figure 4), but genome sequences

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available to date indicate the presence of both enzymes (Derelle which bilins are produced in which algae. Interestingly, several et al., 2006; Palenik et al., 2007; Moreau et al., 2012). Less is known of the dinoflagellates apparently lacking FDBRs are also known about the transition from PebB to HY2, because the PebB/HY2 to be heterotrophic (Jeong et al., 2010), consistent with a con- lineage is apparently absent in extant prasinophyte and chloro- served function for bilin biosynthesis in oxygenic photosynthetic phyte algae. However, there has been a transition in substrate organisms. Bilins themselves do not function as regulators of specificity for this enzyme from 15,16-DHBV (cyanobacterial and gene expression, so they are presumed to act as second messen- rhodophyte PebB) to BV (plant HY2). Biochemical characteriza- gers within a larger signal transduction pathway. Heterotrophic tion of the equivalent enzyme from charophytes may clarify protists could retain the ability to sense bilins but not synthe- this process. It is nevertheless clear that all known members of the size them; this could provide a means of responding to particular Archaeplastida retain at least a single FDBR, but no single FDBR types of prey. We conclude that some type of FDBR is apparently is conserved across all of these lineages. present in all oxygenic photosynthetic lineages, regardless of the A similar picture can be seen upon analyzing FDBRs in algae presence of phytochromes or phycobiliproteins. We next examine that have undergone secondary endosymbiosis (Figure 4), using the known roles of bilins in light and oxygen sensing. recent transcriptomic data (Keeling et al., 2014). Such algae are derived from the capture of a prasinophyte alga (giving rise to A BILIN FOR ALL COLORS: BILINS AS PHYTOCHROME photosynthetic ), a chlorophyte alga (giving rise to chlo- CHROMOPHORES rarachniophytes), or a rhodophyte alga (all other taxa shown in Bilins function as chromophore cofactors for phytochrome pho- Figure 4).Cryptophytealgaeretainphycobiliproteins;therefore, toreceptors (Li and Lagarias, 1992). Phytochromes are reversibly the presence of PebA and PebB in cryptophytes is not surpris- photoswitching photosensory proteins typically containing an N- ing. Cryptophytes of the genus Hemiselmis also contain PcyA terminal photosensory core module (PCM) that autocatalytically (Figure 4), although the significance of this observation is not assembles with bilin to perceive light and a C-terminal domain yet clear. Most (heterokont) algae retain PebA, and homologous to histidine kinases or other signaling domains many retain PebB as well. PebA is apparently the only FDBR (Rockwell and Lagarias, 2010; Auldridge and Forest, 2011). Light present in , whereas PebB is apparently the absorption by the covalently attached bilin chromophore triggers only FDBR retained in (Figure 4). There is still lit- photoisomerization of the bilin 15,16-double bond (Figure 5A). tle data available from chromerids and euglenids, but FDBRs Photoisomerization flips the bilin D-ring relative to the rest appear to be present in these taxa as well. PebA is present in of the chromophore, triggering a series of changes in protein- many dinoflagellates, frequently as the only FDBR detected in chromophore interactions that lead to subsequent structural transcriptomic studies. Biochemical characterization of FDBRs rearrangements, ultimately modulating the signaling state of the from diverse algae will be needed to provide a clearer picture of molecule (Rockwell and Lagarias, 2010; Auldridge and Forest,

FIGURE 5 | Perception of light by phytochrome. (A) Absorption of light by Cys residue. Et, ethyl; P,propionate. (B) In land plants, the two photostates phytochrome triggers reversible photoisomerization of the bilin 15,16-double absorb red (15Z configuration, blue) and far-red (15E configuration, orange) bond, resulting in photoconversion between two photostates. Rings and light. (C) Phytochromes from eukaryotic algae exhibit much more diverse numbering system are indicated for a covalent PCB adduct to a conserved photoperception.

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2011; Song et al., 2011, 2014; Yang et al., 2011). Changes in stramenopile , revealinganunexpectedfar- bilin configuration and protein-chromophore interactions result red/green photocycle. It is thus clear that algal phytochromes in a change in peak absorption upon photoconversion, allow- exhibit greater spectral diversity than land plant phytochromes, ing phytochromes and distantly related cyanobacteriochromes with different proteins able to sense essentially the entire visible (CBCRs) to sense two different colors (Ikeuchi and Ishizuka, spectrum (Figure 5C). 2008; Rockwell and Lagarias, 2010; Auldridge and Forest, 2011; Little is known about the function of phytochromes in algal Rockwell et al., 2011). The 15Z configuration of the bilin is syn- cells, so this remains an open frontier for further investigation. thesized by FDBRs and is typically the dark-stable state. The 15E Recent studies on the prasinophyte alga pusilla indi- photoproduct can rapidly photoconvert back to the 15Z dark state cate that its phytochrome accumulates in the nucleus in light, as upon illumination; many photoproducts are also able to revert do land plant phytochromes (Duanmu et al., 2014). Algae that to the 15Z dark state spontaneously over seconds to days in a lack phytochromes presumably possess other sensory systems or process known as dark reversion (Rockwell et al., 2006). In land adopt lifestyles that no longer require these sensors. Interestingly, plants, the dark state absorbs red light and the photoproduct glaucophytes contain multiple phytochromes (glaucophyte phy- absorbs far-red light, yielding a reversible red/far-red photocycle tochrome sensors or GPS proteins; see Rockwell et al., 2014), so it (Figure 5B). Changes in signaling state upon plant phytochrome is possible that those proteins may have specialized functions. A photoconversion trigger nuclear translocation of phytochrome similar specialization is seen in the distantly related CBCRs. Like and activation of thousands of genes (Hu et al., 2009; Franklin algal phytochromes, different CBCRs provide complete coverage and Quail, 2010). Phytochrome thus acts as a master regulator of the visible spectrum (Ikeuchi and Ishizuka, 2008; Rockwell of developmental processes such as photomorphogenesis and the et al., 2011). Most CBCRs do not yet have known functions, shade avoidance response in land plants (Hu et al., 2009; Franklin but several such proteins have been linked to specialized roles in and Quail, 2010; Casal, 2013). Phytochromes exhibiting similar regulating cyanobacterial photobiology, including and red/far-red photocycles have since been reported from cyanobac- complementary chromatic acclimation (Ikeuchi and Ishizuka, teria, other bacteria, and fungi (Yeh et al., 1997; Davis et al., 2008). The apparent distribution of phytochromes in eukaryotic 1999; Froehlich et al., 2005; Brandt et al., 2008). Those from algae (Figure 4) can also inform examination of phytochrome organisms such as fungi and non-photosynthetic bacteria that function in these organisms: whereas phytochromes are appar- lack FDBRs use biliverdin as chromophore, resulting in a red- ently present in all streptophytes, they are present in only a shifted photocycle (Bhoo et al., 2001; Lamparter et al., 2003, 2004; fraction of prasinophytes and are lost entirely in rhodophytes Froehlich et al., 2005; Brandt et al., 2008). These proteins are and chlorophytes. Hence, it seems unlikely that Archaeplastida implicated in regulating various aspects of photobiology, includ- phytochromes have a single conserved function. A similar argu- ing light harvesting and fungal development (Giraud et al., 2002; ment can be advanced for algae with secondary plastids, based on Röhrig et al., 2013). CBCRs are only known from cyanobacteria transcriptomic data (Keeling et al., 2014). Cryptophytes contain and do not sense far-red light; instead, they exhibit a great vari- multiple phytochromes, again raising the possibility of specialized ety of photocycles spanning the complete visible spectrum and functions. can vary, with some organisms lack- even the near-ultraviolet (Ikeuchi and Ishizuka, 2008; Rockwell ing phytochromes entirely and others containing multiple phy- and Lagarias, 2010; Rockwell et al., 2011). tochromes. There is little information yet available for euglenids, Eukaryotic algal phytochromes have attracted much less chromerids, and for P. chromatophora. Rhodophytes, chloro- attention. Phytochrome from the charophyte alga phytes, haptophytes, dinoflagellates, and chlorarachniophytes caldariorum was shown to use phycocyanobilin (PCB) as the apparently lack phytochrome entirely. The observed sporadic dis- chromophore rather than the phytochromobilin (PB) chro- tribution could indicate that phytochromes are not essential for mophore found in land plants (Wu et al., 1997). This is somewhat many photosynthetic eukaryotes and can be lost. In contrast, surprising in light of more recent transcriptomic and genomic FDBRs are retained and expressed in algal lineages that lack both studies of these algae (Timme et al., 2012; Hori et al., 2014), phytochromes and phycobiliproteins (Figure 4). Hence, it seems because charophyte algae apparently lack the FDBR PcyA that likely that there is a function for bilins in oxygenic photosynthetic synthesizes PCB (Figures 3, 4). PCB synthesis in these algae is eukaryotes independent of known roles for these tetrapyrroles as thus enigmatic. However, the consequences of this chromophore phytochrome and chromophores. change for color perception are minor, and charophyte phy- tochromes exhibit red/far-red photocycles very similar to those LIFE WITHOUT PHYTOCHROME: BILIN-DEPENDENT STRESS of phytochromes from land plants, cyanobacteria, other bacte- RESPONSES IN CHLAMYDOMONAS ria, and fungi (Kidd and Lagarias, 1990; Jorissen et al., 2002). Such widespread conservation of FDBRs implicates additional Much greater spectral diversity has recently been demonstrated biological functions for bilins. This question has been examined in other algal phytochromes, using recombinant expression in in the model chlorophyte Chlamydomonas reinhardtii (Duanmu Escherichia coli cells engineered to express HO and FDBR genes et al., 2013). This study confirmed, using in vitro characteri- (Rockwell et al., 2014). In prasinophyte phytochromes, both zation of recombinant proteins, that Chlamydomonas contains red/far-red and orange/far-red photocycles have been reported. functional HO and FDBR proteins. Cells lacking the HMOX1 Glaucophyte phytochromes exhibit greater diversity still, with gene exhibited pronounced defects in light-dependent chloro- red/blue and blue/far-red photocycles reported to date. This study phyll accumulation and photoautotrophic growth, phenotypes also examined a single phytochrome from a secondary alga, the that could be rescued by addition of exogenous BV to the

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growth media. Such phenotypes could be explained via heme Ochromonas has been evaluated as a possible means of controlling accumulation in HMOX1 mutant cells, resulting in feedback inhi- blooms of toxic cyanobacteria (Van Donk et al., 2009). Cells bition of the trunk pathway common to both heme and chloro- undertaking active feeding may have reduced photosynthetic phyll synthesis (Figure 3). However, expression of a mammalian capacity (Stoecker et al., 1997; Maruyama and Kim, 2013); there- biliverdin reductase in wild-type Chlamydomonas cells mimicked fore, some means of balancing photosynthesis and heterotrophy loss of HMOX1 (Duanmu et al., 2013), indicating that accelerated would be required. This balancing act would be particularly heme turnover does not promote chlorophyll accumulation and important during onset of photosynthesis, when the cell faces an ruling out such an effect. PCB biosynthesis in the Chlamydomonas oxidative stress and has not yet acclimated to a sudden change in plastid was confirmed by plastid expression of a CBCR reporter, the light environment. Similar transient oxidative stresses could and CBCR chromophorylation was shown to be dependent on the also arise during digestion of photosynthetic prey in light, due to presence of a functional copy of the HMOX1 gene, a nuclear gene the presence of chlorophyll in such prey. encoding a plastid-directed HO. The ability to use bilins to respond to oxidative stresses Cells with or without HMOX1 and with or without exogenous associated with photosynthesis and/or the digestion of prey BV were subjected to transcriptomic analysis during dark-light could have been retained to assist in dealing with such chal- transitions (Duanmu et al., 2013). Many genes were induced by lenges. Such a bilin response is consistent with the bilin- light, but these did not correlate with similarly light-responsive mediated regulation of oxidative stress genes in Chlamydomonas. transcripts in plants. Over one hundred genes were regulated Moreover, the importance of transient changes in the light by light in a bilin-dependent manner. Light induction of this environment to eukaryotic algae can be seen in studies of pho- “core group” of genes was suppressed by the addition of exoge- toprotective mechanisms in the diatom Pseudo-nitzschia multis- nous BV, confirming the importance of linear tetrapyrroles in triata, in which photoprotective mechanisms respond not only the process. This core group responding to dark-light transitions to increasing light intensity but also to the rate of increase included members of several gene families implicated in stress (Giovagnetti et al., 2014). Such “rapid response” mechanisms responses and high light responses. In contrast, light-responsive provide protection against sudden changes in the light environ- genes observed during the dark-light transition in land plants ment arising due to vertical mixing phenomena, an unpredictable are heavily biased toward gene families implicated in light har- challenge facing both mixotrophic and photosynthetic marine vesting and chlorophyll synthesis. Hence, the initial response to algae. high light in Chlamydomonas is different from that in land plants, Studies in Chlamydomonas have not unambiguously identified being heavily weighted toward responding to the initial oxidative the actual signaling molecule that is needed to sustain chlorophyll stress occurring at the onset of photosynthesis in the dark-to-light accumulation in the light, because cells lacking PCYA were not transition. available. The formation of carbon monoxide as part of the HO A bilin-based response system is well suited to any stress reaction (Figure 3) raises the possibility of a gaseous second mes- that arises through the combined effects of light and oxygen. senger. However, the phenotypes observed in Chlamydomonas Linear tetrapyrroles can also be produced during the break- cells lacking the HO encoded by HMOX1 are rescued by exoge- down of chlorophyll in plants (Hörtensteiner and Kräutler, 2011; nous BV and can be phenocopied by expression of biliverdin Süssenbacher et al., 2014); therefore a pathway able to detect reductase (Duanmu et al., 2013). These results indicate that the a variety of bilins and related compounds could also report light-dependent response pathway in Chlamydomonas is indeed the presence of chlorophyll. The ability to detect chlorophyll dependent on linear tetrapyrroles rather than on byproducts such could be advantageous for a predator, because chlorophyll is a carbon monoxide or free iron. If BV or chlorophyll-derived break- strongly photodynamic, potentially toxic substance in light. Bilin- down products were to function as signaling molecules, then based responses could also be used to balance photoautotrophic FDBRs would seem unnecessary and the conservation and expres- and heterotrophic metabolism. Direct antioxidant roles for bilins sion of FDBRs in diverse algae would be unexplained. If one (Stocker, 2004) also are possible, but these have not yet been were to assume the existence of a conserved, bilin-based signal- established in photosynthetic organisms. ing pathway for sensing light and oxygen stress, then the sporadic The prasinophyte genera , , distribution of different FDBRs in modern algae would argue , and Micromonas have recently been shown to for a non-conserved signaling molecule, with different bilins fill- exhibit mixotrophy (Maruyama and Kim, 2013; Gast et al., 2014; ing this role in different algae. However, it is also possible that McKie-Krisberg and Sanders, 2014). Phagotrophic engulfment biochemical characterization of more diverse algal FDBRs will of bacterial prey is also well established in secondary algae reveal that many such enzymes have undergone convergent evo- such as chlorarachniophytes, haptophytes, dinoflagellates, and lution to permit synthesis of a common bilin by a range of cryptophytes and in both photosynthetic and heterotrophic phylogenetically diverse FDBRs. Future studies employing a range stramenopiles (Stoecker et al., 1997; Roberts and Laybourn- of disciplines will be necessary to understand the bilin-based Parry, 1999; Moestrup and Sengco, 2001; Adolf et al., 2006; stress pathway or pathways more fully and to test which path- Burkholder et al., 2008; Van Donk et al., 2009; Jeong, 2011; Jeong ways occur in other algae. However, the broad distribution of et al., 2012; Unrein et al., 2014). Hence, the ancestral ability FDBRs in eukaryotic algae and the presence of bilin-based stress to engulf bacteria phagotrophically has remained widespread pathways of Chlamydomonas are consistent with a widespread, during algal evolution. In a number of cases, the bacterial prey bilin-based pathway for detecting the combination of light and can include cyanobacteria; indeed, the mixotrophic stramenopile oxygen stress.

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FUTURE PERSPECTIVES: WHAT CAN BE DONE TO SEE FAR hypothesis. The presence of phytochromes in many algal lineages INTO THE PAST? also indicates that bilins are likely to play multiple roles in algal Primary endosymbiosis clearly was not established “overnight,” photobiology. with an instant transition from free-living predator and prey to obligate endosymbiosis. The Archaeplastida ancestor thus could ACKNOWLEDGMENTS elaborate light- and oxygen-sensing systems during a more grad- This research was supported by grants from the National Science ual transition, and this would have been to its advantage as it Foundation awarded to Debashish Bhattacharya (0625440, adapted from transient oxidative stress during prey digestion to 0936884, 1317114) and from the NIH (R01 GM068552) and prolonged, light-dependent oxidative stress in the presence of USDA National Institute of Food and Agriculture (Hatch project an endosymbiont. Study of intermediate stages in the conver- number CA-D∗-MCB-4126-H) awarded to J. C. Lagarias. We sion from predator/prey to obligate symbiosis would thus be are grateful for the constructive criticisms of three anonymous potentially informative. However, such intermediate stages are no reviewers. longer extant, forcing us to search the amazingly diverse world of protists for analogies. REFERENCES A number of extant organisms are known to graze upon Adl, S. M., Simpson, A. G., Lane, C. E., Lukeš, J., Bass, D., Bowser, S. S., et al. (2012). cyanobacteria and/or eukaryotic algae. Molecular characteriza- The revised classification of eukaryotes. J. Eukaryot. Microbiol. 59, 429–493. doi: tion of such organisms can provide insight into the strategies 10.1111/j.1550-7408.2012.00644.x Adolf, J. E., Stoecker, D. K., and Harding, L. W. Jr. (2006). 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