Sterol Composition and Biosynthetic Genes of Vitrella brassicaformis, a Recently Discovered Chromerid: Comparison to Chromera velia and Phylogenetic Relationship with Apicomplexan Parasites.

Published by the International Society of Protistologists

The Journal of

Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

Sterol Composition and Biosynthetic Genes of Vitrella brassicaformis, a Recently Discovered Chromerid: Comparison to Chromera velia and Phylogenetic Relationship with Apicomplexan Parasites Manoj Khadkaa, Mohamed Salema & Jeffrey D. Leblonda,b a Department of Biology, Middle Tennessee State University, PO Box 60, Murfreesboro, Tennessee 37132 b Ecology and Evolution Group, Middle Tennessee State University, PO Box 60, Murfreesboro, Tennessee 37132

Keywords Chromerida; dinoflagellate; mevalonate; phylogeny; red algae. Correspondence J.D. Leblond, Department of Biology, Middle Tennessee State University, PO Box 60, Murfreesboro, TN 37132, USA Telephone number: +615-898-5205; FAX number: +615-898-5093; e-mail: [email protected] Received: 26 August 2014; revised 4 February 2015; accepted February 5, 2015. doi:10.1111/jeu.12237

ABSTRACT Vitrella brassicaformis is the second discovered species in the Chromerida, and first in the family Vitrellaceae. Chromera velia, the first discovered species, forms an independent photosynthetic lineage with V. brassicaformis, and both are closely related to peridinin-containing dinoflagellates and nonphotosynthetic apicomplexans; both also show phylogenetic closeness with red algal plastids. We have utilized gas chromatography/mass spectrometry to identify two free sterols, 24-ethylcholest-5-en-3b-ol, and a minor unknown sterol which appeared to be a C28:4 compound. We have also used RNA Seq analysis to identify seven genes found in the nonmevalonate/methylerythritol pathway (MEP) for sterol biosynthesis. Subsequent genome analysis of V. brassicaformis showed the presence of two mevalonate (MVA) pathway genes, though the genes were not observed in the transcriptome analysis. Transcripts from four genes (dxr, ispf, ispd, and idi) were selected and translated into proteins to study the phylogenetic relationship of sterol biosynthesis in V. brassicaformis and C. velia to other groups of algae and apicomplexans. On the basis of our genomic and transcriptomic analyses, we hypothesize that the MEP pathway was the primary pathway that apicomplexans used for sterol biosynthesis before they lost their sterol biosynthesis ability, although contribution of the MVA pathway cannot be discounted.

THE phylum Chromerida was first described under the group Alveolata with the discovery of Chromera velia in 2008 (Adl et al. 2012; Moore et al. 2008). The second discovered species, now called Vitrella brassicaformis, was first used as an unnamed chromerid to demonstrate a shared red algal plastid ancestry with C. velia, dinoflagellates, heterokonts, and nonphotosynthetic apicomplexan parasites, which possess a nonphotosynthetic relic plastid termed an apicoplast (Janouskovec et al. 2010). Later the formal description of V. brassicaformis was provided by studying its morphology, ultrastructure, and life cycle, and it was formally classified under the phylum Chromerida and family Vitrellaceae, whereas C. velia was classified in the family Chromeraceae (Obornık et al. 2012). Despite placement in different families, phylogenetic analysis of eight concatenated nuclear genes has shown that

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V. brassicaformis and C. velia are both close relatives of apicomplexan parasites (Janou skovec et al. 2010). Comparatively, C. velia and V. brassicaformis possess a combination of similar, as well as different features, thus illustrating that even though they are in the same phylum, there are distinct differences between these only two chromerids named to date. For example, morphological and genetic features, such as tubular mitochondrial cristae, cortical alveoli, subpellicular microtubules, heterodynamic flagella, terminally tapered flagella, micronemes, and a bacterial Rubisco gene, are found in both chromerids, whereas a pseudoconoid, chromerosome, finger-like projection on the shorter flagellum, and four-celled sporangia are only found in C. velia but not in V. brassicaformis (Obornık et al. 2011, 2012). Vitrella brassicaformis also possesses multiple cells containing sporangia, a multiple-laminated cell

© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 786–798

Vitrella brassicaformis Sterols

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wall, pyrenoid, and compacted plastid genome that are absent in C. velia (Obornık et al. 2012). Sterols are isoprenoid-derived, amphipathic, ringed lipids that are commonly biosynthesized de novo in many eukaryotes via either a classical acetate/mevalonate pathway (MVA) and/or a more recently discovered nonmevalonate/ methylerythritol pathway (MEP) (Bloch 1965; Eisenreich et al. 2004; Nes 2011). Sterols perform several essential functions, such as regulating membrane dynamics that affect cell proliferation and differentiation, displaying hormonal activity in plants, and acting as an effector molecule by modulating function of membrane-bound proteins such as H+-ATPase (Dufourc 2008; Grunwald 1971; Hartmann 1998; Nes 2011). As a result of their prevalence in eukaryotes, sterols have also been utilized as molecular biomarkers to trace algal productivity and to make chemotaxonomic inference for classifying various algal groups (Moldowan et al. 1990; Rampen et al. 2010). Most eukaryotes synthesize sterols de novo, however, parasitic apicomplexans have lost this ability (Labaied et al. 2011), and no other sterol biosynthetic genes have been detected downstream of the biosynthetic intermediate, isopentenyl diphosphate, except farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase (Artz et al. 2011; Jord~ao et al. 2013; Ling et al. 2007), the product of which is utilized in protein prenylation (van der Meer and Hirsch 2012). However, genes related to the MEP pathway have been detected in apicomplexans (Cassera et al. 2004; Clastre et al. 2007), and parasitic apicomplexans have shown the ability to scavenge sterols from a host cell in the form of cholesteryl esters (Coppens 2006; Coppens et al. 2000; Nishikawa et al. 2005). Vitrella brassicaformis and C. velia, being closely related to apicomplexans (Janouskovec et al. 2010), can be utilized to speculate how sterols were biosynthesized in apicomplexans before the biosynthetic machinery was lost. A recent study on the sterol composition of C. velia showed the presence of 24R-methylcholesta-5,22Edien-3b-ol, 24R-ethylcholesta-5,22E-dien-3b-ol, and 24-ethylcholest-5-en-3b-ol as major sterols; these three sterols are common in higher plants and seemingly unrelated algae such as chlorarachniophytes (Leblond et al. 2005) and glaucocystophytes (Leblond et al. 2011). Referring to the Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway database (Ogata et al. 1999), there are six and seven enzymatic steps that lead to the synthesis of isopentenyl diphosphate via the MVA and MEP pathways, respectively. Both the pathways utilize different precursor molecules to synthesize this C5 isoprene unit. Once formed, isopentenyl diphosphate is isomerized to form dimethylallyl diphosphate, which is used to form geranyl diphosphate. The product repetitively adds isopentenyl diphosphate to form farnesyl diphosphate and geranyl geranyl diphosphate. Two molecules of farnesyl diphosphate can further be condensed to squalene. Squalene is then oxidized to squalene 2,3-oxide by squalene epoxidase. Oxidosqualene cyclase acts on squalene 2,3-oxide to convert generally into one of two cyclic forms, 4,4,14-trimethyl-5acholesta-3,24-dien-3b-ol (lanosterol) and 4,4,14a-trimethyl5a-9,19-cyclocholesta-24-en-3b-ol (cycloartenol), depending

on the organism (as reviewed by Abe et al. 1993; Benveniste 1986; Ramos-Valdivia et al. 1997). Various sterols are then synthesized via steps following this bifurcation into the lanosterol and cycloartenol pathways, respectively. Three sterol biosynthetic genes, namely smt1, fdft1, and idi1, were discovered in C. velia after examination of expressed sequence tag data (Leblond et al. 2012). However, further study of the sterol composition and biosynthetic genes in V. brassicaformis and C. velia (using the more powerful RNA Seq technique) can be used to better decipher sterol biosynthesis in these two chromerids, in addition to identifying chemotaxonomic and phylogenetic relationships between themselves, and possibly dinoflagellates and apicomplexans. Chromerids are the closest photosynthetic and autotrophic relative to apicomplexans (Janou skovec et al. 2010; Moore et al. 2008), and although apicomplexans have lost their photosynthetic capabilities and capacity to synthesize sterols de novo (Labaied et al. 2011), they possess an active MEP pathway (Cassera et al. 2004) that functions in the biosynthesis of various isoprene molecules such as dolichol, carotenoids, ubiquinone, and others (as review by Odom 2011). Therefore, we conducted this study with the following objectives: (1) to determine the sterol composition of V. brassicaformis and compare it to the previously determined sterol composition of C. velia (and other algal groups, such as dinoflagellates), (2) to identify (via genome and transcriptome analyses) genes related to sterol biosynthesis in V. brassicaformis with the goal of identifying usage of the MVA and/or MEP pathway(s) for sterol biosynthesis, and (3) to make phylogenetic comparisons of the translated protein sequences with other algae, such as dinoflagellates, and parasitic apicomplexans. To this end, we present this first characterization of the sterols and related biosynthetic genes of V. brassicaformis. MATERIALS AND METHODS Culture and lipid extraction Vitrella brassicaformis NCMA 3155 (also known as CCMP 3155) was procured from the Provasoli-Guillard National Center for Marine Algae and Microbiota (West Boothbay Harbor, ME, USA). The cultures were grown autotrophically in triplicate in 1 liter of L1 medium (Guillard and Hargraves 1993) at room temperature under a 14/10 h light/dark cycle at an irradiance of approximately 50 lmol photon/m2/s, and harvested during late exponential phase at a density of approximately 1.2 9 105 cells/ml by filtration onto a precombusted 934-AH Whatman glass fiber filter (GE Healthcare Bio-Sciences, Pittsburgh, PA). The filtered algal biomass was subjected to extraction and total amount of lipid was determined. Lipids were extracted according to the technique described by Leblond and Chapman (2000). Processing and analysis of sterols The total lipid extract was separated into five fractions via column chromatography using 1 g of 100–200 mesh Unisil


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silica (Clarkson Chromatography, South Williamsport, PA) activated at 120 °C for 4 h. The following solvent regime was used to separate lipids according to polarity (Leblond and Chapman 2000): (1) 12 ml methylene chloride (sterol esters), (2) 15 ml 5% acetone in methylene chloride with 0.05% acetic acid (free sterols, tri-and diacylglycerols, and free fatty acids), (3) 10 ml 20% acetone in methylene chloride (monoacylglycerols), (4) 45 ml acetone (glycolipids), and (5) 15 ml methanol with 0.1% acetic acid (polar lipids, including phospholipids and betaine lipids). Fraction two containing free sterols were saponified according to the techniques described by Leblond and Chapman (2002). After saponification, sterols were derivatized with 0.5 ml N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylethylchlorosilane at 80 °C for 0.5 h. The reagent was evaporated under a stream of nitrogen and the trimethylsilyl (TMS) derivatives redissolved in 20 ll of 1:1 hexane/methyl-tert-butyl ether. Gas chromatography/mass spectrometry (GC/MS) was used to analyze the derivatized sterols in the positive-ion electron impact (EI) mode using the following conditions: 1 ll injected via splitless injection with injector set at 280 °C, transfer line set at 275 °C, helium carrier at 28 cm/s, 70 eV with a scanning range of 50–600 amu, and a cycle time of 1.1 s using a Thermo TSQ Quantum GC/MS (Austin, TX) and a DB-5 column (30 m 9 0.25 lm film thickness; J&W Scientific Incorporated, Folsom, CA). The GC temperature was 50 °C for 1 min, 50–170 °C at 15 °C/ min, 170–300 °C at 10 °C/min with a hold of 11 min. Sterols were also analyzed using positive-ion chemical ionization (CI) using the same GC conditions and the mass spectrometry conditions specified by Dahmen and Leblond (2013); this was done to obtain better fragmentation in the high mass range. Relative retention times (RRT) to cholest-5-en-3b-ol (cholesterol) as its TMS derivative were calculated according to the methodology of Jones et al. (1994). Examination of uninoculated L1 medium as a negative control using the same filtering and lipid processing techniques as described above has shown an absence of sterols (data not shown). Identification of sterol biosynthetic genes using genome and transcriptome data The Sequence Read Archive (SRA) database from National Center for Biotechnology and Information (NCBI, Leinonen et al. 2011; Wheeler et al. 2008) was utilized to retrieve transcriptome data for V. brassicaformis NCMA 3155. The data can be accessed using SRA accession number SRX215482 or using the following link: http:// www.ncbi.nlm.nih.gov/sra?term=(SRX215482)%20NOT% 20cluster_dbgap%5BPROP%5D. The short transcripts were de novo assembled to 50,755 contigs using CLC Genomics Workbench (version 6.0.2) and mapped to the KEGG automatic annotation server (KAAS) (Moriya et al. 2007) for the identification of sterol biosynthetic genes. The KAAS mapping identified 12 genes related to sterol biosynthesis that included the genes related to an isoprenoid biosynthetic pathway. The transcriptome data for

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C. velia were accessed from NCBI-SRA database (Leinonen et al. 2011a; Wheeler et al. 2008) using the link http:// www.ncbi.nlm.nih.gov/sra/SRX090189[accn] and accession number SRX090189. All the assembled transcripts were further utilized to identify the genes related to sterol and isoprenoid biosynthetic pathway using KAAS. The method of genome analysis for V. brassicaformis is described in Material S1. Phylogenetic analyses of sterol biosynthetic genes The gene sequences for 1-D-deoxy-xylulose phosphate reductoisomerase (dxr), 2-C-methyl- D-erythritol 2, 4-cyclodiphospahte synthase (ispF), 2-C-methyl-D-erythritol 4phosphate cytidylyltransferase (ispD), and isopentenyl diphosphate isomerase (idi) were identified from the transcriptome analyses of V. brassicaformis and C. velia, and were utilized to predict the protein sequence using an online tool called the genescan web server at MIT (Burge and Karlin 1998). The parameters were set up as organisms: Arabidopsis; suboptimal exon cutoff: 1; print option: predicted CDS and peptides. The FASTA-formatted protein sequences for the apicomplexans, dinoflagellates, diatoms, and plants were searched using gene names in the NCBI database (Sayers et al. 2009). The assembled transcripts, along with the NCBI-retrieved protein sequences, were utilized for the construction of maximum likelihood trees for four enzymes leading to formation of isopentenyl diphosphate: 1-D-deoxy-xylulose phosphate reductoisomerase (DXR), 2-C-methyl-D-erythritol 2, 4-cyclodiphospahte synthase (ISPF), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ISPD), and isopentenyl diphosphate isomerase (IDI). All the protein sequences were MUSCLE (Edgar 2004) aligned using the Molecular Evolutionary Genetics Analysis (MEGA) 6.0 program (Tamura et al. 2013), and converted to PHYLIP format using Mesquite version 2.75 (Maddison and Maddison 2011). The aligned protein sequences were analyzed to find the best model fit using ProtTEST 3.3 software (Darriba et al. 2011). The best model was chosen and used in Randomized Accelerated Maximum Likelihood (RAxML) (Stamatakis 2014) for the construction of phylogenetic trees that was installed in Cyberinfrastructure for Phylogenetic Research (CIPRES) gateway version 3.3 (Miller et al. 2010). RESULTS Sterol profile of Vitrella brassicaformis Fraction two of the total lipid extract was derivatized and analyzed via GC/MS to examine the free sterol profile of V. brassicaformis. The analysis displayed two sterols, with the more abundant of the two being identified as 24-ethylcholest-5-en-3b-ol at m/z 486 (Fig. 1, side chain stereochemistry not specified); this sterol had a retention time of 42.54 min, RRT of 1.13 relative to cholesterol, and relative abundance of approximately 83.3 2%. The lesser sterol was found to be an unknown, apparently C28:4 sterol at m/z 466 (Fig. 2), retention time of 37.65 min, RRT of



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0.29 relative to cholesterol and relative abundance of approximately 16.7 2%. The identified sterol was confirmed by comparing to the spectrum of a 24-ethylcholest5-en-3b-ol (sitosterol) standard, which showed a fragmentation pattern of M+ – TMS-O-H – side chain (m/z 255), M+ – TMS-O – H (m/z 396), and M+ – CH3 (m/z 471; Fig. 1). 24-Ethylcholest-5-en-3b-ol was found to be present at approximately 9 pg/cell, and the unknown sterol was found to be present at approximately 2 pg/cell; these amounts are similar to what has been observed in the dinoflagellate, Pfiesteria piscicida, and other dinoflagellates (Leblond and Chapman 2004, and references therein). Phylogenetic analysis using protein sequences for the dxr, ispF, ispD, and idi genes Phylogenetic analyses were performed using translated transcripts of four genes related to isoprenoid and sterol biosynthesis. The relationship was constructed using plants, red, and green algae, diatoms, dinoflagellates (both

peridinin-containing and one, Lepidodinium chlorophorum, with an aberrant plastid), and apicomplexans, and other groups of algae wherever the data were available. The phylogenetic analyses of four protein sequences related to isoprenoid and sterol biosynthetic pathway clearly differentiates the protists under study into two groups with green and red algal-derived plastids. A maximum likelihood tree for protein sequence for the dxr gene (Fig. 3) showed V. brassicaformis grouped with the peridinin-containing dinoflagellate, Pyrocystis lunula, with a bootstrap support of 100. Vitrella brassicaformis and P. lunula further grouped with the green-pigmented dinoflagellate, L. chlorophorum (bootstrap support 100), and all three species grouped with the (proto)dinoflagellate Oxyrrhis marina with a bootstrap support of 91. The dinoflagellates and V. brassicaformis were closely grouped with perkinsids and then to apicomplexans, suggesting V. brassicaformis is more closely related to apicomplexans than C. velia. As the heterokonts, apicomplexans, and peridinin-containing dinoflagellates used in this analysis consist of red algal-derived plastids,


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the grouping of the two chromerids with these organisms further reinforces their red algal origin. The clustering of the two chromerids within specific phylogenetic groups is further discussed below. Vitrella brassicaformis formed a sister clade with C. velia in the analyses of the ISPF protein sequence with bootstrap support of 77 (Fig. 4), and the ISPD protein sequence (Fig. 5) showed Nannochloropsis gaditana, a heterokont, as a close relative to V. brassicaformis (bootstrap support 75), and Guillardia theta, a cryptophyte, to C. velia (bootstrap support 47). In addition, V. brassicaformis and C. velia formed a sister clade in the maximum likelihood tree constructed using the IDI sequence, where both were closely related to N. gaditana (bootstrap support of 53; Fig. 6). For all the analyses above, the two chromerids consistently grouped with the heterokonts, apicomplexans, and rhodophytes. In summary, the four maximum likelihood trees generated using isoprenoid biosynthetic genes demonstrated rhodophytes as a possible ancestor of sterol biosynthetic genes in V. brassicaformis and C. velia.

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DISCUSSION Although chromerids and apicomplexans are closely related to each other (Janou skovec et al. 2010; Moore et al. 2008), they have adopted vastly different modes of life, with the former being photosynthetic and the latter being obligately pathogenic. A direct comparison of sterol composition cannot be made between apicomplexans and chromerids because apicomplexans do not synthesize sterols de novo (Coppens 2006), although they do possess the ability to synthesize other isoprenoid compounds (Guggisberg et al. 2014) and to convert choles et al. 2013; Nishikawa terol to cholesteryl esters (Botte et al. 2005). Dinoflagellates, a closely related group to both chromerids and apicomplexans as based on ultrastructural and phylogenetic features (Janou skovec et al. 2010; Moore et al. 2008; Obornık et al. 2012), however, synthesize a wide variety of sterols de novo (Patterson 1971, 1991), which does not generally include those produced by C. velia and V. brassicaformis (see discussion by Leblond et al. 2012).



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A

Figure 3 Maximum likelihood tree generated by using RAxML with LG+G model for 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) protein sequence. Section A of this figure is magnified for clarity.


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Figure 4 Maximum likelihood tree generated by using RAxML with LG+G model for 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ISPF) protein sequence.

24-Ethylcholest-5-en-3b-ol is the predominant sterol in both V. brassicaformis and C. velia; however, C. velia produces an additional array of sterols, namely 24-methylcholesta-5,22E-dien-3b-ol and 24-ethylcholesta-5,22E-dien3b-ol, that are not found in V. brassicaformis but are

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commonly found in plant and other groups of algae but not in dinoflagellates (Leblond et al. 2012). Based on the KEGG reference pathway (Ogata et al. 1999) for the terpenoid biosynthesis in Arabidopsis thaliana, it takes approximately 10 steps to biosynthesize 24R-ethylcholest-



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Figure 5 Maximum likelihood tree generated by using RAxML with LG+G model for 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ISPD) protein sequence.

5-en-3b-ol (sitosterol) after biosynthesis of 4,4,14a-trimethyl-5a-9,19-cyclocholesta-24-en-3b-ol (cycloartenol); 24R-ethylcholesta-5,22E-dien-3b-ol (stigmasterol) is formed by a subsequent desaturation of the side chain of 24R-ethylcholest-5-en-3b-ol. As V. brassicaformis does not produce 24R-ethylcholesta-5,22E-dien-3b-ol under these growth conditions, it is possible that it lacks the enzyme to introduce this additional unsaturation. It should also be noted that V.

brassicaformis did not produce the 24-methylcholesta5,22E-dien-3b-ol produced by C. velia, thus indicating additional missing steps required for the formation of this sterol from the earlier intermediate 24-methylenelophenol (4a-methyl-5a-ergosta-7,24-dien-3b-ol; see discussion below). Sterols can be attributed to particular classes, families and sometimes even species of algae, thus making them


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Figure 6 Maximum likelihood tree generated by using RAxML with LG+G model for isopentenyl diphosphate isomerase (IDI) protein sequence.

potentially useful for chemotaxonomic comparisons (Patterson 1991). For example, Karenia brevis, produces two predominant sterols, (24R)-4a-methyl-5a-ergosta-8(14),22dien-3b-ol (gymnodinosterol) and 27-nor-(24R)-4a-methyl5a-ergosta-8(14),22-dien-3b-ol (brevesterol), that are not produced by most other dinoflagellates (Giner et al. 2003; Leblond and Chapman 2002). Rhodophytes, the predicted ancestor of chromerid plastids, typically produce cholesterol and several other sterols, such 5a-cholestan-3b-ol (cholestanol), cholesta-5,24-diene-3b-ol (desmosterol),

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cholest-5,7-dien-3b-ol (dehydrocholesterol) (Patterson 1991), that are different from the sterols produced by V. brassicaformis and C. velia. However, several classes of algae, such as glaucocystophytes (Leblond et al. 2011), chlorophytes (as reviewed by Volkman 1986), chlorarachniophytes (Leblond et al. 2005), have been reported to produce 24-ethylcholest-5-en-3b-ol. The MEP pathway (Rohmer et al. 1996) is commonly found in eubacteria, green algae, and the chloroplasts of higher plants (Kuzuyama 2002; Lohr et al. 2012), although a more comprehen-



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sive survey of sterol-producing organisms is needed, and utilizes glyceraldehyde 3-phosphate and pyruvate as precursors to synthesize isopentenyl diphosphate. Per the genes found in this study, the following steps are relevant (enzymes abbreviation typed bold in parentheses for each step): (1) Glyceraldehyde 3-phosphate and pyruvate are condensed to 1-deoxy-D-xylulose-5-phopsphate (DOXP) by DOXP synthase (DXS); (2) DOXP is converted to 2-Cmethyl-D-erythritol-4-phosphate (MEP) by DOXP-reductoisomerase (DXR); (3) MEP is converted to cytidine 50 -diphosphate-2-C-methyl-D-erythritol (CDP-ME) by CDPME-synthase or 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ISPD); (4) CDP-ME kinase (ISPE) catalyzes formation of CDP-methyl-D-eryhritol-2-phosphate (CDP-ME-2P) from CDP-ME; (5) CDP-ME-2P is converted into 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (ME-2, 4-cPP) by 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (ME-cPP) synthase (ISPF); (6) 4-hydroxy-3-methyl-2-(E)butenyl-diphosphate (HMBPP) is synthesized from ME2,4-cPP by HMBPP synthase (ISPG); and (7) ultimately isopentenyl diphosphate is synthesized from HMBPP by catalytic action of HMBPP reductase (ISPH) (Lichtenthaler 2010). Isopentenyl diphosphate is converted to an isomer, dimethylallyl diphosphate, by the enzyme isopentenyl diphosphate isomerase (IDI). The transcriptome analysis of V. brassicaformis identified all the seven genes related to the MEP pathway (Material S2) and two genes related to the MVA pathway (Material S3) suggesting that sterol biosynthesis in V. brassicaformis may involve both the MVA and MEP pathways. Beyond this series of early steps, Benveniste (1986) divides the overall pathway of sterol biosynthesis into four basic steps: (1) the lanosterol–cycloartenol bifurcation, (2) the alkylation reaction that adds methyl and ethyl groups at the C-24 position of sterol, (3) opening of the cyclopropane ring of cycloeucalenol and obtusifoliol, and (4) the later stage characterized by removal of methyl groups at the C-4 and C-14 position of sterol, and conversion of D8 into D5 sterols. The lanosterol pathway is common in nonphotosynthetic eukaryotes, such as animals and fungi, whereas the cycloartenol pathway is commonly found in photosynthetic eukaryotes, including algae, bryophytes, and tracheophytes (Benveniste 1986). Two sterol methyltransferases, SMT1 and SMT2, act on cycloartenol and 24-methylenelophenol, respectively, to add an ethyl group at the C-24 position to synthesize sitosterol and eventually et al. stigmasterol in plants (Bloch 1983; Bouvier-Nave 1998; Nes 2000); in this pathway, 24-methylcholesta5,22E-dien-3b-ol (campesterol) can be a substrate for side chain methylation to form sitosterol. Lohr et al. (2012) present a tabular summary for the presence of MVA and MEP pathways in various groups of algae and protists, among which the apicomplexa and dinoflagellates possess the MEP pathway, whereas other groups of algae such as the rhodophytes, glaucocystophytes, streptophytes, euglenophytes, chlorarachniophytes, heterokontophytes, haptophytes, and cryptophytes possess both the pathways. The genome and transcriptome analyses of V. brassicaformis also showed genes related to both the cycloartenol and

lanosterol pathways (data not shown). Genes such as lanosterol synthase (lss) and cycloartenol synthase (cas) that are specific for lanosterol and cycloartenol pathways, respectively, were found along with additional genes like delta(14)-sterol reductase (erg24, lanosterol pathway), methyl sterol monooxygenase (erg25, lanosterol pathway), and sterol 24-C-methyltransferase (smt1, cycloartenol pathway). Protein sequences (ISPF, ISPD, ISPC, and IDI) related to isoprenoid biosynthesis found in the transcriptomes of both C. velia and V. brassicaformis were utilized for generating maximum likelihood trees with bootstrap support to determine the evolutionary relationship as it relates to sterol biosynthesis between C. velia and V. brassicaformis, and these two chromerids with other groups of algae. According to the chromalveolate hypothesis (CavalierSmith 1999), four major eukaryotic lineages-cryptophytes, haptophytes, heterokonts, and alveolates—that contain chlorophyll c originated from a single endosymbiotic event between the ancestors of those groups and a red alga(e). Several phylogenetic studies have however challenged this hypothesis to suggest multiple endosymbiotic events in chromalveolates (Baurain et al. 2010); these are evidenced by the occurrence of ancient recruited green algalderived genes in diatoms and other chromalveolates (Frommolt et al. 2008; Moustafa et al. 2009). Phylogenetic analyses of various genes such as signal recognition docking protein, folate biopterin transporter, vitamin K epoxide reductase, and fructose-bisphosphate aldolase in C. velia, have shown sharing of both red and green algal-derived genes (Burki et al. 2012; Woehle et al. 2011). Thus, in performing gene-by-gene analyses of isoprenoid biosynthetic genes in V. brassicaformis, it can be determined if there are any green algal-derived genes in the isoprenoid biosynthetic pathway (i.e. MVA and/or MEP) as seen in C. velia and other chromalveolates. Maximum likelihood analysis of the DXR protein sequence showed V. brassicaformis to be more closely related to apicomplexans than to C. velia, and P. lunula to be the closest relative of V. brassicaformis; conversely, red algae were more closely related to C. velia; this result is consistent with the recent study on small subunit rRNA phylogeny, which suggests V. brassicaformis to be more closely related to apicomplexans than is C. velia (Gile and Slamovits 2014). Thus, the phylogeny concluded that for the DXR protein sequence, the two chromerids were not grouped together, but fell within a large group of organisms containing the red algae and those with red algal-derived plastids (Fig. 3). Maximum likelihood analysis using the ISPD protein sequence (Fig. 5) showed N. gaditana as the closest relative of V. brassicaformis, and G. theta as the closest relative of C. velia, whereas V. brassicaformis and C. velia grouped with N. gaditana using the IDI protein sequence (Fig. 6). Guillardia theta has been shown to share plastid ancestry with red algae (Douglas and Penny 1999), and several genes in N. gaditana showed homology with both red and green algae (Radakovits et al. 2012). The phylogenetic grouping of V. brassicaformis with N. gaditana is consistent with the


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finding of Obornık et al. (2012) that suggests morphological similarity and identical pigmentation between V. brassicaformis and eustigmatophytes. Based on the maximum likelihood tree generated using the DXR protein sequence, it can be said that V. brassicaformis is the closest photosynthetic relative to apicomplexans; however, the sterol composition of V. brassicaformis cannot be compared directly with apicomplexans because of their inability to synthesize sterols de novo. As the phylogenetic analyses suggest the red algal origin of sterol biosynthesis in V. brassicaformis, it can be expected that the sterol composition of rhodophytes and V. brassicaformis should be similar unless changed by an event such as horizontal gene transfer. Indeed, the sterol compositions of rhodophytes and chromerids do not match with each other, suggesting a fundamental difference in at least some reactions of their respective biosynthetic pathways. It can be suggested that the genes related to the MEP pathway were red algal-derived, however, the genes that function beyond isopentenyl diphosphate might have been transferred to V. brassicaformis horizontally from another algal group, such as glaucocystophytes or chlorophytes, so as to biosynthesize sterols which are found in those groups of algae. Horizontal gene transfer in chromerids is not unprecedented. For example, phylogenetic inference for a ferrochelatase gene has suggested a horizontal gene transfer event in the ancestor of C. velia (and apicomplexans) from a proteobacterium (Koreny et al. 2011). In addition, previous phylogenetic studies of several nuclear genes in C. velia have revealed their green as well as red algal origin (Burki et al. 2012; Woehle et al. 2011). In conclusion, C. velia produces a larger diversity of sterols than V. brassicaformis, and like C. velia, 24-ethylcholest-5-en-3b-ol produced by V. brassicaformis is also found in other groups of algae and higher plants and thus cannot be of biomarker utility. The transcriptome (Material S2) and genome analyses (Material S3) of V. brassicaformis showed the existence of MVA as well as MEP pathways suggesting sterol biosynthesis occurring via either of the pathways; however, we observed more genes transcribed from the MEP pathway. Phylogenetic analyses of isoprenoid biosynthetic genes (leading to sterol biosynthesis) in V. brassicaformis and C. velia showed their red algal origin. As the Chromerida are closely related to apicomplexans (Janouskovec et al. 2010; Moore et al. 2008), based on our finding on sterol composition and biosynthetic genes, we put forward the hypothesis that apicomplexans might have synthesized sterols utilizing primarily the MEP pathway before their sterol biosynthetic pathway was lost. However, it remains an open question, due to loss of genes late in the sterol biosynthesis pathway, as to whether the sterols which may have been produced by apicomplexans in the past would have resembled those of chromerids (and green algae, for example) or those of red algae. The genome analysis of V. brassicaformis identified two genes, hydroxymethylglutaryl-CoA synthase (hmgcs), and hydroxymethylglutaryl-CoA reductase (hmgcr), related to the MVA pathway. The finding of MVA pathway genes

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in V. brassicaformis, a close relative of apicomplexans may help to date the loss of the MVA pathway in extant apicomplexans.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Material S1. Method for Vitrella brassicaformis genome analysis. Material S2. KEGG pathway map showing MEP pathway genes in Vitrella brassicaformis genes (genes were identified utilizing transcriptome data available in Sequence Read Archive Database; accession number SRX215482). Material S3. KEGG pathway map showing MVA pathway genes identified in Vitrella brassicaformis (genes were identified utilizing genome data available in Sequence Read Archive Database, accession number SRX152523).


The Organellar Genomes of Chromera and Vitrella, the Phototrophic Relatives of Apicomplexan Parasites.

Increased growth and pigment content of Chromera velia in mixotrophic culture.

Novel type of red-shifted chlorophyll a antenna complex from Chromera velia. I. Physiological relevance and functional connection to photosystems.

Isoprenoid metabolism in apicomplexan parasites.

Transcript maturation in apicomplexan parasites.

InsP3 Signaling in Apicomplexan Parasites.

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites.

Oxidative stress control by apicomplexan parasites.

Recent advances in understanding apicomplexan parasites.

ATG8 localization in apicomplexan parasites: apicoplast and more?

Recently discovered adipokines and cardio-metabolic comorbidities in childhood obesity.

Sterol composition of butters and margarines.

Integration of RNA-seq and proteomics data with genomics for improved genome annotation in Apicomplexan parasites.

Phylogenetic relationship of Hepatozoon blood parasites found in snakes from Africa, America and Asia.

Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites.

Host cell invasion by apicomplexan parasites: the junction conundrum.

Phylogenetic comparison of avian haemosporidian parasites from resident and migratory birds in northern Japan.

Protein trafficking in apicomplexan parasites: crossing the vacuolar Rubicon.

Found in translation: functions and evolution of a recently discovered alternative proteome.

Biological Activity of Recently Discovered Halogenated Marine Natural Products.

The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus.

The effect of nitrogen limitation on acetyl-CoA carboxylase expression and fatty acid content in Chromera velia and Isochrysis aff. galbana (TISO).

Members of a recently discovered subfamily of cytokinin receptors display differences and similarities to their classical counterparts.

Riproximin is a recently discovered type II ribosome inactivating protein with potential for treating cancer.