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. Author manuscript; available in PMC: 2018 Oct 1.
Published in final edited form as: J Plant Physiol. 2017 May 31;217:57–67. doi: 10.1016/j.jplph.2017.05.022

Ferredoxin-dependent bilin reductases in eukaryotic algae: ubiquity and diversity

Nathan C Rockwell 1, J Clark Lagarias 1,*
PMCID: PMC5603387  NIHMSID: NIHMS882008  PMID: 28641882

Abstract

Linear tetrapyrroles (bilins) are produced from heme by heme oxygenase, usually forming biliverdin IXα (BV). Fungi and bacteria use BV as chromophore for phytochrome photoreceptors. Oxygenic photosynthetic organisms use BV as a substrate for ferredoxin-dependent bilin reductases (FDBRs), enzymes that produce diverse reduced bilins used as light-harvesting pigments in phycobiliproteins and as photoactive photoreceptor chromophores. Bilin biosynthesis is essential for phototrophic growth in Chlamydomonas reinhardtii despite the absence of phytochromes or phycobiliproteins in this organism, raising the possibility that bilins are more generally required for phototrophic growth by algae. We here leverage the recent expansion in available algal transcriptomes, cyanobacterial genomes, and environmental metagenomes to analyze the distribution and diversification of FDBRs. With the possible exception of euglenids, FDBRs are present in all photosynthetic eukaryotic lineages. Phylogenetic analysis demonstrates that algal FDBRs belong to the three previously recognized FDBR lineages. Our studies provide new insights into FDBR evolution and diversification.

Keywords: algal evolution, biliverdin, bioinformatics, cyanobacteriochromes, oxygenic photosynthesis, phycocyanobilin, phycoerythrobilin, phylogenetics, phytochromobilin, secondary endosymbiosis, tetrapyrrole metabolism

INTRODUCTION

Tetrapyrroles are required for diverse metabolic processes essential to terrestrial life. Linear tetrapyrroles derived from heme (bilins) play important roles in the biology of photosynthetic organisms. Bilins are synthesized from heme via heme oxygenase (HO) to yield biliverdin IXα (BV, Fig. 1). In oxygenic photosynthetic organisms, BV is a substrate for ferredoxin-dependent bilin reductases (FDBRs: (Dammeyer and Frankenberg-Dinkel, 2008)) that generate a range of bilins by reducing different double bonds (Fig. 1). Cyanobacteria contain as many as three FDBRs: PcyA, PebA, and PebB. Phylogenetic analysis places these three enzymes into distinct lineages (Rockwell et al., 2017). Eukaryotic FDBRs characterized to date also belong to these three lineages. For example, HY2 enzymes from streptophytes are part of the PebB lineage, whereas PCYA from chlorophyte algae belongs to the PcyA lineage. PUBS from prasinophyte and streptophyte algae and nonvascular plants belongs to the PebA lineage (Rockwell et al., 2017). Thus, photosynthetic organisms can contain multiple FDBRs producing different reduced bilins, sometimes referred to as phycobilins or phytobilins.

Figure 1. Bilin biosynthesis by ferredoxin-dependent bilin reductases (FDBRs).

Figure 1

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After initial breakdown of heme by heme oxygenase (encoded by the HY1 gene in streptophytes) to yield biliverdin IXα (BV), different FDBRs produce different bilins. PcyA and HY2 from streptophyte algae reduce BV to phycocyanobilin (PCB) in a 4-electron reduction, whereas HY2 from land plants reduces BV to phytochromobilin (PΦB) in a 2-electron reduction. PebA and PebB reduce BV to phycoerythrobilin (PEB) in a 2-enzyme, 4-electron reduction. The same reaction can be carried out by PebS or PcyX as 1-enzyme reactions. PUBS from Viridiplantae reduces BV to phycourobilin (PUB) in a 4-electron reduction. The chromophoric conjugated π systems of different bilins are colored, approximately matching their apparent visual colors. Orange, moieties whose structure differs in different bilins. P, propionate.

The bilins produced by FDBRs play important roles in the photobiology of oxygenic photosynthetic organisms. Phycobilin chromophores are used by light-harvesting phycobiliproteins in cyanobacteria and eukaryotic glaucophyte, rhodophyte, and cryptophyte algae, frequently as components of large phycobilisome antennae. Phycobilisome-deficient mutant strains of Synechococcus sp. PCC 7002 are viable (Alvey et al., 2011). Land plants use phytochromobilin (PΦB, Fig. 1) as the chromophore for phytochrome photoreceptors. Plant phytochromes are critical master regulators for many processes, but plants lacking phytochrome are also viable (Hu et al., 2013). FDBRs would thus not be expected to be essential for oxygenic photosynthesis.

Surprisingly, there is evidence that FDBRs are essential for phototrophic growth. Mutant strains of Synechococcus sp. PCC 7002 lacking PcyA could only be obtained in the presence of heterologously expressed HY2 from Arabidopsis thaliana (Alvey et al., 2011). PcyA and HY2 produce structurally related phycocyanobilin (PCB) and PΦB, respectively (Fig. 1). There is also evidence that bilin biosynthesis is essential in the chlorophyte alga Chlamydomonas reinhardtii (Duanmu et al., 2013), which lacks phytochromes and phycobiliproteins but retains HO and PCYA. A mutant strain lacking HMOX1, encoding plastid HO, was unable to grow phototrophically. Addition of exogenous BV afforded partial rescue of hmox1Δ phenotypes (Duanmu et al., 2013). These results thus implicate essential functions for bilins in cyanobacteria and green algae.

A requirement for bilins in oxygenic photosynthesis would seem surprising given the intense research into this process for over a century. However, a requirement for bilins in oxygenic photosynthesis implies the presence of HO and one or more FDBRs in all oxygenic photosynthetic organisms, including diverse algae (Fig. 2). Preliminary BLAST searches of algal transcriptomes (Keeling et al., 2014; Matasci et al., 2014) confirmed the presence of FDBRs in many algal lineages. In the current work, we use phylogenetic analysis to examine the distribution and diversification of FDBRs in photosynthetic organisms. The results confirm the presence of FDBRs in all algal lineages, with the possible exception of photosynthetic euglenids, and confirm that eukaryotic FDBRs belong to the three known lineages. Our studies provide new insight into FDBR loss, duplication, and diversification.

Figure 2. Evolution of photosynthetic eukaryotes.

Figure 2

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A simplified view of the tree of life is shown, based on recent studies and assuming a monophyletic Archaeplastida for simplicity (Burki et al., 2016; Price et al., 2012; Spang et al., 2015). Primary (dashed blue lines), secondary (thin solid lines), and tertiary (thick grey lines) endosymbioses are shown. Oxygenic photosynthetic organisms are color-coded by light-harvesting strategy and plastid ancestry. Only endosymbioses resulting in creation of a photosynthetic, carbon-fixing organelle are shown.

METHODS

Construction of FDBR sequence alignments and phylogenies

Additional bacterial and phage FDBR sequences were identified using BLAST (Altschul et al., 1997) searches of the DOE-IMG database and of the Genbank protein and GSS (genomic survey sequences) databases. Eukaryotic FDBR sequences were identified using BLAST searches of the Genbank protein, EST (expressed sequence tag), TSA (transcriptome shotgun assembly), and SRA (sequence read archive) databases, of publicly available OneKP (one thousand plants, (Matasci et al., 2014)) transcriptomes, and of transcriptomes generated by the Marine Microeukaryotes Transcriptome Sequencing Project (MMETSP, (Keeling et al., 2014)). In some cases, including EST and SRA data, multiple hits were combined to yield a complete FDBR sequence using the approach we previously applied to PCYA from Cyanophora paradoxa CCMP329 (Rockwell et al., 2017). Accession information for FDBR sequences used in this paper is provided in Supplemental Table 1.

Sequence alignments constructed with MAFFT (Katoh et al., 2002) were used to identify FDBR sequences with complete or nearly complete catalytic core regions, and an in-house script was used to remove positions with ≥5% gaps and to add secondary structure and solvent accessibility calculated using DSSP (Kabsch and Sander, 1983). Positions that adopted different structural environments in different FDBR crystal structures were assigned as exposed/other for use with the EX_EHO substitution model (Le and Gascuel, 2010). Each resulting alignment was used to infer a structurally informed ML phylogeny using PhyML-structure (Le and Gascuel, 2010) with the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) to assess statistical support (Anisimova and Gascuel, 2006). Command-line settings for MAFFT were (--genafpair --maxiterate 16 --clustalout --reorder). Command-line settings for PhyML-structure were (-m EX_EHO -M PART -a e -c 4 -v e -o tlr). Structures with the PDB accessions 2D1E, 2G18, 2X9O, and 2VCK were used as references. In analyzing phylogenetic relationships within the three FDBR lineages, statistical support is reported in the figures using SH-aLRT for the individual tree and in the text using the percent recovery for the 64 trees that were manually analyzed in lieu of conventional bootstrap analysis.

RESULTS AND DISCUSSION

Alignment and phylogenetic analysis of FDBRs

We used our previous phylogenetic analysis of HY2 from streptophyte algae (Rockwell et al., 2017) as a starting point for constructing new alignments including both eukaryotic sequences and sequences from more diverse bacteria. Alignments were then used to infer structurally informed ML phylogenies using PhyML-structure (Le and Gascuel, 2010). We initially generated 80 trees. These trees had a range of 186–365 sequences and 154–179 characters after removal of gap-enriched positions. Alignments with fewer characters typically included incomplete sequences. We tested two possible outgroups: CPO sequences from cyanobacteria and RCCR sequences from cyanobacteria and land plants. Both of these enzymes share the overall fold of FDBRs, despite limited sequence similarity, and hence provide suitable outgroups for structurally informed phylogenetic analysis (Hagiwara et al., 2006; Sugishima et al., 2009). The RCCR-based outgroup gave more consistent root placement and is similar to that used in our previous FDBR phylogeny (Rockwell et al., 2017).

We next focused on sequences with complete or nearly complete coverage of the catalytic core region, generating 64 trees from alignments having 189–219 sequences and 186–207 characters after removal of gap-enriched positions. These 64 trees were examined manually at nodes of interest in lieu of conventional bootstrap analysis to avoid misleading results caused by inclusion of particular sequences. The most common topology in these 64 trees (47%) recovered a monophyletic PcyA lineage diverging first relative to the outgroup, with monophyletic PebA and PebB lineages diverging from each other subsequently (Fig. 3). Initial divergence of a monophyletic PebA lineage followed by separation of monophyletic PcyA and PebB lineages was observed in 22% of trees (Fig. 3). These results illustrate the current uncertainty in our understanding of how the three lineages arose during FDBR evolution: the three lineages are probably monophyletic, but we have not reconstructed the relationships among them. The behavior of FDBRs within the three lineages was more robustly recovered. Representative views of these lineages (Fig. 4–6) thus provide new insight into the distribution and diversification of FDBRs during evolution.

Figure 3. Phylogenetic analysis of FDBR evolution.

Figure 3

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Divergence of the three FDBR lineages relative to the RCCR outgroup is shown for the two topologies most frequently recovered in the final set of 64 phylogenies.

Figure 4. Diversification of the PcyA lineage.

Figure 4

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A detailed view of a phylogenetic analysis of members of the PcyA lineage is shown, with sequences color-coded by origin. Circles denote SH-aLRT confidence (black, SH-aLRT ≥0.9; white, 0.9 >SH-aLRT ≥0.7. Experimentally characterized FDBRs are in bold.

Figure 6. Diversification of the PebB lineage.

Figure 6

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A detailed view of a phylogenetic analysis of members of the PebB lineage is shown, with sequences color-coded by origin. Circles denote SH-aLRT confidence (black, SH-aLRT ≥0.9; white, 0.9 >SH-aLRT ≥0.7. Experimentally characterized FDBRs are in bold.

FDBRs in bacteria and phages

PcyA is ubiquitous in photosynthetic cyanobacteria and is thought to be essential in Synechococcus sp. PCC 7002 (Alvey et al., 2011), whereas PebA and PebB are specifically present in organisms that synthesize phycoerythrobilin (Dammeyer and Frankenberg-Dinkel, 2008). FDBR sequences are absent in the genomes of nonphotosynthetic Melainabacteria, sister to cyanobacteria (Soo et al., 2014), but present in the genomes of Candidatus Atelocyanobacterium spp. (also known as UCYN cyanobacteria), photoheterotrophs derived from unicellular nitrogen-fixing photosynthetic cyanobacteria (Bombar et al., 2014). Ca. Atelocyanobacterium contained only PcyA, and these sequences were robustly associated with other cyanobacterial PcyA sequences (90% support; Fig. 4). We also found examples of all three FDBRs in the recently reported genomes of cyanobacterial sponge symbionts (Ca. Synechococcus spongiarum: (Burgsdorf et al., 2015)). It is well established that marine cyanobacteria with greatly reduced genomes belonging to the genera Cyanobium, Synechococcus, and Prochlorococcus (hereafter, the CSP clade) form a monophyletic group connected to other cyanobacteria by a characteristically long branch (Shih et al., 2013). Cyanobacterial sponge symbionts have recently been shown to be sister to the CSP clade (Burgsdorf et al., 2015), and indeed sponge symbiont PebA and PebB sequences were sister to PebA and PebB sequences from the CSP clade (100% support; Figs. 5–6).

Figure 5. Diversification of the PebA lineage.

Figure 5

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A detailed view of a phylogenetic analysis of members of the PebA lineage is shown, with sequences color-coded by origin. Circles denote SH-aLRT confidence (black, SH-aLRT ≥0.9; white, 0.9 >SH-aLRT ≥0.7. Experimentally characterized FDBRs are in bold.

PcyA sequences from Ca. S. spongiarum and from the CSP clade also clustered with phage PcyA sequences, metagenomic sequences from marine and brackish environments, PCYA sequences from photosynthetic Paulinella spp., and PCYA sequences from photosynthetic euglenids (Fig. 4). We were able to define two groups within this cluster. One group comprised PcyA from CSP cyanobacteria, PCYA from Paulinella spp., PcyA from phages, and PcyA from metagenomic samples. The second comprised PcyA from Ca. S. spongiarum and PCYA from euglenids. The (sponge + euglenid) PcyA lineage was sister to (CSP + Paulinella + phage + metagenome) lineage (67% support), and phage PcyA sequences were derived from CSP PcyA sequences (72% support). These two lineages together formed a monophyletic clade (the expanded CSP PcyA clade) that was not robustly related to PcyA sequences from other cyanobacteria.

In addition to PcyA, phage genomes can also encode PebS or PcyX (Dammeyer et al., 2008; Ledermann et al., 2016). PebS and PcyX both catalyze the 4-electron reduction of BV to PEB, but PebS belongs to the PebA lineage and PcyX belongs to the PcyA lineage (Figs. 4–5). PebS sequences formed a monophyletic clade most often (56% support) placed as sister to a clade of FDBRs from phaeophyte algae (phaeophyte PEBZ; Fig. 5 and see below). PcyX sequences also formed a monophyletic clade derived not from cyanobacterial FDBRs but from PcyA sequences from other bacteria, consistent with previous work (Ledermann et al., 2016).

FDBRs from the nonoxygenic photosynthetic bacterium Bradyrhizobium sp. ORS278 and the metagenomically sequenced actinobacterium SCGC AAA041-L13 were previously recovered as sister to phage PcyX sequences (Jaubert et al., 2007; Ledermann et al., 2016). Bradyrhizobium PcyA produces PCB (Jaubert et al., 2007), so the transition from bacterial PcyA to phage PcyX is a change in regiospecificity. We observed an additional closely related FDBR in the genome of Roseomonas stagni DSM 19981 (Rhodospirillales, α-proteobacteria). In both Bradyrhizobium sp. ORS278 and R. stagni, the pcyA gene was associated with a phytochrome gene, a heme oxygenase gene, and a large cluster of gas vesicle genes (Fig. S1). The FDBR from actinobacterium SCGC AAA041-L13 is also adjacent to a heme oxygenase, but phytochrome and gas vesicle genes are apparently absent (Fig. S1). The sequences from Bradyrhizobium, actinobacterium SCGC AAA041-L13, and R. stagni were recovered as part of a paraphyletic group that were the closest relatives to phage PcyX (Fig. 4), as expected (Ledermann et al., 2016).

We also observed two additional cases of possible FDBRs in nonphotosynthetic bacteria. In the first case, both available genomes for Chondromyces spp. (Myxococcales, δ-proteobacteria) encode possible FDBRs. These sequences were typically recovered as sister to the PcyA lineage (52% support) or sister to a combined (PebA + PebB) lineage (30% support, Fig. 7). These sequences could thus belong to a fourth FDBR lineage. Chondromcyes FDBRs are adjacent not to HO genes but to genes implicated in corrin metabolism and signal transduction. The function of these proteins is thus unclear. FDBRs may also be present in δ-proteobacterium DG_8 and members of Candidatus Schekmanbacteria, all known only from environmental metagenome sequencing. BLAST searches identified FDBRs as the closest known relatives to these sequences, but this relationship was distant. These sequences were most frequently recovered (63%) as sister to all FDBRs in phylogenetic analyses. We were unable to find cognate heme oxygenases in the available genomes for Ca. Schekmanbacteria and δ-proteobacterium DG_8. We therefore consider these proteins to be less promising FDBR candidates and chose to place the arbitrary root of the phylogeny between (RCCR + δ-proteobacterium DG_8/Ca. Schekmanbacteria sequences) and all other sequences (Fig. 7).

Figure 7. Phylogenetic analysis of possible novel FDBR lineages.

Figure 7

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The most frequently observed topology for overall FDBR evolution (Fig. 3) is shown with the most likely placements of candidate FDBRs from Chondromyces spp., Ca. Schekmanbacteria, and δ-proteobacterium DG_8. Percentages indicate support for placement of Chondromyces sequences. This placement of sequences from Ca. Schekmanbacteria and δ-proteobacterium DG_8 was observed in 63% of phylogenies.

Eukaryotic FDBRs and photosynthesis

BLAST searches identified candidate FDBRs in transcriptomes and/or genomes of every photosynthetic eukaryotic taxon for which data are available. FDBRs were present in 90% of examined algal transcriptomes, and phylogenetic analysis placed all eukaryotic FDBR sequences within the three previously recognized FDBR lineages (Figs. 4–6). Cyanobacterial FDBRs were not found to be basal to eukaryotic sequences in any lineage, indicating that cyanobacterial sequences have diverged significantly since primary endosymbiosis gave rise to Archaeplastida.

The absence of an FDBR gene in a given transcriptome does not preclude the presence of such a gene in the underlying genome. To date, the only photosynthetic eukaryotic genomes lacking annotated FDBRs are those of Aureococcus anophagefferens CCMP1794 and Chrysochromulina sp. CCMP291. Strikingly, reexamination of the archived sequence reads for both genomes revealed partial FDBR sequences (Fig. S2). For A. anophagefferens, the C-terminus of the candidate FDBR sequence could also be identified in transcriptomes. For Chrysochromulina sp. CCMP291, a closely related FDBR was observed in the transcriptome of C. rotalis UIO044 (Keeling et al., 2014), confirming that FDBRs are transcribed in Chrysochromulina spp. It thus seems plausible that FDBRs are ubiquitous in photosynthetic eukaryotes.

FDBRs are rare in nonphotosynthetic eukaryotes. FDBRs are absent in fungi, and metazoans, and the previously photosynthetic apicomplexans. The cryptophyte genus Cryptomonas includes both photosynthetic and nonphotosynthetic strains (Hoef-Emden, 2005). Analysis of transcriptomes relvealed multiple FDBRs in photosynthetic Cryptomonas curvata CCAP 979/52 but not in nonphotosynthetic C. paramecium CCAP 977/2a. Cryptophytes are members of the Cryptista, and FDBRs were not detected in transcriptomes for the nonphotosynthetic Cryptistans Roombia truncata (a katablepharid), Goniomonas spp., and Palpitomonas bilix (Burki et al., 2016).

Surprisingly, we were able to detect a candidate FDBR sequence in the transcriptome for the nonphotosynthetic chlorophyte Polytomella parva SAG 63-3 (Keeling et al., 2014). This sequence was not closely related to PCYA sequences from photosynthetic chlorophytes and was excluded from the detailed phylogenetic analysis. However, incomplete closely related sequences could be found in other Polytomella transcriptomes (Fig. S3). P. parva SAG 63-3 also retained a candidate ortholog for at least one of the two heme oxygenases found in the C. reinhardtii genome. It is thus possible that Polytomella spp. have retained a complete phycobilin biosynthesis pathway.

Loss and diversification of FDBRs in Archaeplastida

A single primary endosymbiosis has been proposed to give rise to the Archaeplastida supergroup, comprising glaucophytes, rhodophytes, and Viridiplantae (Price et al., 2012), although monophyly of the Archaeplastida is still under debate (Burki et al., 2016). Glaucophytes retain phycobilisomes (Price et al., 2012). PCYA genes were found in all glaucophyte transcriptomes and genomes. These PCYA sequences were sister to a subset of extant cyanobacterial PcyA sequences (100% support, Fig. 4).

Extant rhodophytes also retain phycobiliproteins for light harvesting. PEBA and PEBB genes were found in all rhodophytes examined, with the exception of Cyanidioschyzon merolae. C. merolae belongs to the early-diverging Cyanidiales; its genome encodes a PCYA gene (Fig. 4). This finding is in contrast to the presence of PEBA and PEBB genes in the genome of another member of the Cyanidiales, Galdieria sulphuraria (Figs. 5–6). PCYA from C. merolae was recovered as sister to cyanobacterial PcyA sequences but was not robustly associated with glaucophyte PCYA enzymes (Fig. 4). Interestingly, PCYA sequences from the cryptophyte algal genera Chroomonas and Hemiselmis formed a monophyletic group descended from C. merolae PCYA with modest (73%) support.

Rhodophyte PEBA sequences formed a paraphyletic group that may be ancestral to a monophyletic group of cryptophyte PEBA sequences (80% support; Fig. 5 and see below). Rhodophyte PEBB enzymes also formed a paraphyletic group that was clearly ancestral to cryptophyte PEBB sequences (100% support; Fig. 6 and see below). In both cases, (rhodophyte + cryptophyte) sequences were closely related to cyanobacterial sequences (86% support for PEBA and 88% support for PEBB).

The third Archaeplastida lineage is the Viridiplantae, comprising the streptophyte lineage and the prasinophyte/chlorophyte lineage (Duanmu et al., 2014; Leliaert et al., 2016). PCYA genes were found in prasinophyte and chlorophyte algae but not in streptophytes. Similarly, HY2 genes were only found in streptophytes. Prasinophyte/chlorophyte PCYA sequences and streptophyte HY2 sequences each formed a monophyletic clade with 75% support (Figs. 4 & 6). The PebA lineage is represented by PUBS in both Viridiplantae lineages (Fig. 5). PUBS sequences from the two Viridiplantae lineages do not form a monophyletic clade. Candidate PUBS sequences from Mesostigma viride strains were not closely related to other PUBS sequences (Fig. S4), and complete PUBS sequences could not be obtained for other early-diverging streptophytes. It thus may prove possible that a Viridiplantae PUBS clade will be established in the future.

We also observed an additional FDBR in transcriptomes from the prasinophyte genera Tetraselmis and Pyramimonas, which we designate PCYZ. Phylogenetic analysis most often (61% support) placed these sequences sister to a clade comprising phage PcyX and non-cyanobacterial PcyA (Fig. 4). Key catalytic residues in cyanobacterial PcyA (Tu et al., 2007) and eukaryotic PCYA (Table 1) were not conserved in this group. Interestingly, we were unable to detect PUBS in Tetraselmis and Pyramimonas, raising the possibility that PCYZ has functionally replaced PUBS in these prasinophyte genera. Multiple strains clearly contained both PCYA and PCYZ sequences (Fig. 4).

Table 1.

Potential catalytic residues of FDBRs1

Taxon Enzyme H71 E73 H85 D102 D217
cyanobacteria PcyA H E H D D
glaucophytes PCYA H E H D D
Cyanidioschyzon merolae PCYA H E H D D
cryptophytes PCYA H E H D D/E
euglenids PCYA H E H D D
photosynthetic Paulinella PCYA H E H D D
P. ovalis PCYA H E H D E
prasinophyte/chlorophyte PCYA H E H D varies
prasinophyte/chlorophyte PCYZ T/P V/A L E K
bacteria PcyA H E/D H D E/D/P
phage PcyA H E H D D/K
phage PcyX H D H D P
phage PebS R A/V/T N D D
phaeophytes PEBZ R S S S E
diatoms PEBX R T R G E
prasinophytes PUBS R T N D D
dinoflagellates PUBS R T/S N D/E D
streptophyte PUBS R T N D D
chlorarachniophytes PEBA1 R E N D D
chlorarachniophytes PEBA2 R T N N D
chromerids PEBA R T N D D
stramenopile PEBA R T/L N D D
haptophytes PEBA R T N D D
cyanobacteria PebA R T/S N D D
cryptophytes PEBA R T N D D
rhodophytes PEBA R T N D D
cyanobacteria PebB R A N D D
rhodophytes PEBB R A N D D
cryptophytes PEBB R A/C N D D
streptophytes HY2 R C/M/L/A N/D D/N D
haptophytes, Hemiselmis PEBB R A/L N D D
stramenopiles PEBB R A/L/C N D D
Chondromyces spp. FDBR R V N D D
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1

Based on mutagenesis studies reported in (Tu et al., 2007).

FDBRs in Paulinella species

A second primary endosymbiosis has occurred in some species of the testate Rhizarian amoeba Paulinella (Lhee et al., 2017). Photosynthetic Paulinella species acquired photosynthesis and carbon fixation via endosymbiosis of a member of the CSP clade. The three available Paulinella plastid genomes contain PCYA genes, although two of the protein sequences were identical (Fig. S5A). Paulinella PCYA sequences belonged to the expanded CSP clade (Fig. 4), consistent with the evolutionary history of these algae.

Single-cell genome sequencing of six cells of the nonphotosynthetic P. ovalis has been reported (Bhattacharya et al., 2012). Sequencing reads from one of these cells include a possible FDBR sequence (Fig. S5B). This sequence was associated with Bradyrhizobium PcyA and related bacterial sequences (83% support; Fig. 4). BLAST searches did not detect related sequences for the other five cells. It remains unclear whether this is sequence is a bona fide FDBR from P. ovalis or instead is a contaminating sequence from a marine bacterium.

FDBRs in chlorarachniophytes

Chlorarachniophytes, another Rhizarian lineage, acquired photosynthesis via endosymbiosis of a chlorophyte alga (Curtis et al., 2012), predicting the presence of PCYA in chlorarachniophytes. However, available chlorarachniophyte transcriptomes contain 1–2 sequences belonging to the PebA lineage (Fig. 5). Two FDBRs are present in the genome of Bigelowiella natans CCMP2755 (Curtis et al., 2012), implicating a similar situation in other chlorarachniophytes. Several chlorarachniophyte transcriptomes contained multiple candidate FDBRs. Two groups can be distinguished by examination of candidate catalytic residues (Table 1), but the available complete sequences are too inabundant to conclude that there are two chlorarachniophyte FDBRs.

FDBRs in photosynthetic euglenids

Some euglenids have acquired photosynthesis via endosymbiosis of a prasinophyte alga related to extant Pyramimonadales (Turmel et al., 2009). Euglenids belong to the Excavata, an early-diverging eukaryotic supergroup (Burki et al., 2016). A prasinophyte plastid ancestor would be expected to contain PCYA, and we identified PCYA sequences in euglenid transcriptomes from Euglena gracilis and Eutreptiella isolates (Ebenezer et al., 2017; Keeling et al., 2014).

Euglenid PCYA sequences belonged to the expanded CSP clade (Fig. 4). This result could indicate that these FDBR sequences are associated with bacterial contaminants. However, closely related sequences are found in all three transcriptomes, so it is possible that these euglenid FDBR sequences could be encoded by previously undetected cyanobacterial symbionts or via acquisition of cyanobacterial FDBR sequences via HGT. Further studies will be required to resolve this question.

FDBRs in haptophytes

Haptophyte algae acquired photosynthesis via secondary endosymbiosis of a rhodophyte alga. Recent work established haptophytes as sister to the nonphotosynthetic centrohelids in the Haptista (Burki et al., 2016), with (Haptista + SAR) sister to (Cryptista + Archaeplastida). We found FDBR sequences in haptophyte transcriptomes but not in centrohelid transcriptomes (Burki et al., 2016; Keeling et al., 2014). Most haptophyte FDBRs were part of a clade belonging to the PebB lineage that also included PEBB sequences from the cryptophyte genus Hemiselmis (Fig. 6). This relationship did not hold for other cryptophyte PEBB sequences.

Two haptophyte FDBR sequences from Pavlova sp. CCMP459 and from the unclassified prymnesiophyte CCMP2436 were associated with the PebA lineage (Fig. 5). This difference could arise due to variable levels of gene expression and/or transcriptomic coverage. However, a PebA-related gene could not be readily detected in the genome sequence reads for Chrysochromulina sp. CCMP291. It is possible that there are differences in tetrapyrrole and bilin biology among haptophytes.

FDBRs in stramenopiles

Stramenopiles (also known as heterokonts) are a diverse eukaryotic group including both nonphotosynthetic and photosynthetic lineages. Together with alveolates and rhizarians, they form a monophyletic assemblage known as the SAR clade (Burki et al., 2016). A stramenopile clade, the Ochrophyta, is proposed to include all photosynthetic species (Derelle et al., 2016; Sevcikova et al., 2015). Such ochrophytes acquired photosynthesis via endosymbiosis of a eukaryotic rhodophyte alga. One would thus expect the presence of PEBA and PEBB genes, but not PCYA genes, in ochrophytes.

As expected, PCYA sequences were not detected in ochrophytes. PEBA genes were widespread. The diatoms Corethron pennatum L29A3 and Proboscia alata PI_D3 apparently had multiple PEBA transcripts (Fig. 5), although it is not clear whether this arises due to alternative splicing or gene duplication. PEBB sequences were less abundant but were still found in diatoms, phaeophytes, and other ochrophyte lineages (Fig. 6). It is currently unclear whether the relative inabundance of stramenopile PEBB sequences arises due to lower expression levels or loss of the PEBB gene. Stramenopile PEBA sequences were not monophyletic, but stramenopile PEBB sequences were monophyletic with modest support (75%).

We also observed two cases in which stramenopile PEBA genes had apparently duplicated and diverged, potentially giving rise to new FDBRs (Fig. 5). In one case, a subset of diatom sequences were shown to form a small clade most frequently recovered (58%) as descended from diatom PEBA sequences. These sequences also had characteristic differences at potential catalytic residues (Table 1), leading us to designate these sequences as diatom PEBX (Fig. 5). In the second case, a group of phaeophyte FDBR sequences formed a clade most frequently recovered as sister to phage PebS sequences (56% support; Fig. 5). Potential catalytic residues in this clade were distinct from those of stramenopile PEBA sequences and diatom PEBX sequences, so we designate these sequences as phaeophyte PEBZ (Table 1).

FDBRs in chromerids

Chromerid algae (Alveolata) are the closest known photosynthetic relatives of the formerly photosynthetic apicomplexans. Two chromerids, Chromera velia CCMP2878 and Vitrella brassicaformis CCMP3155, have been characterized genomically and transcriptomically (Woo et al., 2015). Chromerids are thought to have acquired photosynthesis via secondary endosymbiosis of a rhodophyte alga or via tertiary endosymbiosis of an ancient ochrophyte (Sevcikova et al., 2015). In either case, one would expect to find PEBA and/or PEBB in chromerids. Both C. velia and V. brassicaformis contain candidate PEBA sequences (Fig. 5). C. velia encodes 2 PEBA genes, whereas V. brassicaformis encodes only one. Genomes are available for both organisms, so the difference in the number of FDBR genes is likely to be reliable. However, it is not currently clear whether this difference is significant. C. velia and V. brassicaformis are not closely related, and additional chromerid diversity may exist (Janouskovec et al., 2012). Future studies may thus provide additional information.

FDBRs in dinoflagellates

Dinoflagellates (Alveolata) are among the most abundant organisms in surface marine waters (Janouskovec et al., 2017). Photosynthetic dinoflagellates initially acquired photosynthesis via endosymbiosis of a rhodophyte, but the resulting secondary plastid has been replaced in some lineages. Dinoflagellates such as Karenia brevis and Karlodinium micrum carried out tertiary endosymbiosis of a haptophyte, whereas the dinotoms Durinskia baltica CSIRO CS-38, Kryptoperidinium foliaceum CCMP 1326, and Glenodinium foliaceum CCAP 1116/3 carried out tertiary endosymbiosis of a diatom. At least one dinoflagellate, Noctiluca scintillans, can live photosynthetically with the assistance of a chlorophyte endosymbiont or can live heterotrophically without it. Other dinoflagellates transiently acquire photosynthesis via kleptoplasty, as in Dinophysis acuminata. Phylotranscriptomic studies implicate both N. scintillans and D. acuminata as having lost functional red-derived plastids in the past (Janouskovec et al., 2017).

We did not observe FDBR sequences in transcriptomes for heterotrophic dinoflagellates such as Oxyrrhis marina CCMP1795 and Crypthecodinium cohnii Seligo, organisms which retain nonphotosynthetic plastids. FDBR sequences were readily detected in transcriptomes for photosynthetic dinoflagellates. Most such sequences formed a clade in the PebA lineage (Fig. 5), with one such sequence per dinoflagellate transcriptome. Gymnodinium catenatum GC744 contained two such sequences, both belonging to the core dinoflagellate FDBR clade. FDBR sequences from the dinotoms D. baltica and K. foliaceum were instead grouped with PEBA and PEBB sequences from diatoms or ochrophytes (Figs. 5–6) and hence are likely to originate in the diatom endosymbiont. Core dinoflagellate FDBRs were recovered as descended from (69% support) or sister to (30% support) prasinophyte PUBS sequences, so we designate them as PUBS rather than PEBA (Fig. 5).

We also observed FDBR sequences in the nonphotosynthetic N. scintillans and D. acuminata. The candidate FDBR sequence in N. scintillans belonged to the core dinoflagellate PUBS clade. The two FDBR sequences in D. acuminata were closely related to those from the cryptophyte alga Geminigera cryophila CCMP2564 (Fig. S6). Phylogenetic analysis clearly placed the D. acuminata PEBB gene with cryptophyte PEBB sequences (Fig. 6). Plastids from G. cryophila are first acquired via kleptoplasty by the ciliate Myrionecta rubra, which in turn is subject to kleptoplasty by D. acuminata (Wisecaver and Hackett, 2010). FDBR genes are not encoded on cryptophyte plastid genomes reported to date, and D. acuminata FDBR sequences are not identical to those from G. cryophila (Fig. S6). It seems likely that D. acuminata FDBR sequences are derived from cryptophyte prey but incorporated into the D. acuminata nuclear genome, as for the cryptophyte-derived gene for PsbM (photosystem II subunit M) in the nuclear genome of D. acuminata (Wisecaver and Hackett, 2010).

FDBRs in cryptophytes

Recent work has shown that cryptophytes are members of the Cryptista, along with nonphotosynthetic katablepharids, goniomonads, and P. bilix (Burki et al., 2016). Cryptophytes acquired photosynthesis via secondary endosymbiosis of a rhodophyte alga. One would therefore expect cryptophyte algae to contain PEBA and PEBB. The model cryptophyte Guillardia theta CCMP2712 is known to contain a functional PEBB enzyme and a putative PEBA enzyme (Overkamp et al., 2014). The presence of PCYA in the extremophilic rhodophyte C. merolae could also implicate the presence of PCYA in cryptophytes.

PEBA was ubiquitous in photosynthetic cryptophytes but absent in at least one nonphotosynthetic isolate of Cryptomonas (see above). Cryptophyte PEBA sequences were closely related to rhodophyte PEBA sequences (Fig. 5). PEBB was also widespread in photosynthetic cryptophytes. However, cryptophyte PEBB sequences were not monophyletic: PEBB sequences from the genus Hemiselmis were part of a clade also including haptophyte PEBB sequences, whereas other cryptophyte PEBB sequences were descended from rhodophyte PEBB sequences as expected (Fig. 6). Uniquely, PEBB from Chroomonas mesostigmatica CCMP1168 was present as a tandem fusion with a PEBA gene (Fig. S7). Both C. mesostigmatica PEBA sequences were part of the cryptophyte PEBA clade, and C. mesostigmatica PEBB was part of the cryptophyte PEBB clade (Figs. 5–6).

Transcriptomes from C. mesostigmatica and Hemiselmis spp. also contained PCYA sequences not found in other cryptophytes. These sequences were recovered as a monophyletic clade (Fig. 4) that was most often (73%) recovered as descended from the rhodophyte PCYA sequence from C. merolae. The evolutionary relationship between PCYA enzymes in cryptophytes and in C. merolae raises the possibility that the ancestral rhodophyte giving rise to the cryptophyte plastid retained all three cyanobacterial FDBRs, with most modern rhodophytes losing PCYA genes at a later date. Characterization of additional members of the Cyanidiales will be important to address this ambiguity.

Future research on bilin biosynthesis

Even the great expansion in available data for eukaryotic algae has left us with an incomplete picture of several key transitions in algal evolution. We have very few PCYA and PUBS sequences from early-diverging prasinophyte algae, and even fewer PUBS sequences from early-diverging streptophyte algae. We also do not have any information on FDBRs from Palmophyllales (Leliaert et al., 2016). Transcriptomic or genomic data are available for only a few chromerids, glaucophytes, and euglenids, and FDBR sequences have not been reported at all for more recently described secondarily photosynthetic lineages (Choi et al., 2017; Janouskovec et al., 2012). It will thus be interesting to revisit this analysis as more data become available.

Our work highlights a disparity between the FDBRs that have been experimentally characterized and the actual diversity of known FDBRs. To date, approximately sixteen FDBRs have been characterized using in vitro enzymology and/or heterologous reconstitution of bilin biosynthesis. Of these enzymes, only one was found in a secondarily photosynthetic alga (Overkamp et al., 2014). Moreover, recent work has shown that closely related FDBRs can carry out different reactions (Rockwell et al., 2017). Our current study provides a starting point in remedying this deficiency by illustrating the extent to which FDBR diversity remains undersampled experimentally.

We have also identified several potentially new FDBRs in algae. Diatom PEBX is apparently a duplication of an ancestral PEBA gene, but changes in potential catalytic residues (Table 1) implicate a different enzymatic function. Phaeophyte PEBZ presents a similar case, but PEBZ is not robustly derived from phaeophyte PEBA sequences and its origins are less clear. The presence of PCYZ only in Pyramimonas and Tetraselmis spp. raises several questions. These genera are not closely related (Duanmu et al., 2014), and PUBS could not be detected in transcriptomes for either genus. This situation could arise through loss of PCYZ or PUBS after early emergence of PCYZ in prasinophytes or through later emergence of PCYZ in one of these two genera with subsequent transfer to the other.

Our studies support the hypothesis that bilin biosynthesis is ubiquitous in oxygenic photosynthetic organisms. With the possible exception of euglenids, all photosynthetic eukaryotes apparently contain FDBRs. Euglenid PCYA sequences clade with cyanobacterial PcyA sequences in the expanded CSP clade and hence may arise from bacterial contaminants or symbionts. However, these sequences could also have been acquired from a cyanobacterial genome independently of plastid acquisition. It is even possible that a cyanobacterial symbiont could supply bilin to euglenids in trans during phototrophic growth. Our studies provide a new palette of FDBRs for understanding the diversity and ubiquity of bilins in photosynthetic eukaryotes and presage similar analysis of heme oxygenase, the other enzyme in the bilin biosynthesis pathway.

Supplementary Material

supplement

Acknowledgments

This work was funded by NIH grant R01 GM068552 (to J. C. L.). The authors thank Kevin Rockwell for assistance in constructing M. viride PUBS sequences from SRA data.

Footnotes

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