J. Phycol. 48, 768–783 (2012) Ó 2012 Phycological Society of America DOI: 10.1111/j.1529-8817.2012.01169.x MOLECULAR EVOLUTION OF GLUTAMINE SYNTHETASE II AND III IN THE CHROMALVEOLATES1 Sohini Ghoshroy and Deborah L. Robertson2 Biology Department, Clark University, 950, Main Street, Worcester, MA 01610, USA Glutamine synthetase (GS) is encoded by three glutamine synthetase III; molecular evolution; distinct gene families (GSI, GSII, and GSIII) that are Rhodophytes broadly distributed among the three domains of life. Abbreviations: BPP, Bayesian posterior probabil- Previous studies established that GSII and GSIII ity; EGT, endosymbiotic gene transfer; HGT, isoenzymes were expressed in diatoms; however, less horizontal gene transfer; JGI ⁄ DOE, Department is known about the distribution and evolution of the of Energy Joint Genome Institute; MLBS, maxi- gene families in other chromalveolate lineages. mum-likelihood bootstrap support; SP, signal Thus, GSII cDNA sequences were isolated from peptide; TP, transit peptide three cryptophytes (Guillardia theta D. R. A. Hill et Wetherbee, Cryptomonas phaseolus Skuja, and Pyreno- monas helgolandii Santore), and GSIII was sequenced from G. theta. Red algal GSII sequences were Marine phytoplankton plays a major role in glo- obtained from Bangia atropurpurea (Mertens ex bal primary productivity and biogeochemical cycles. Roth) C. Agardh; Compsopogon caeruleus (Balbis ex Several lineages within the Chromalveolata super- C. Agardh) Mont.; Flintiella sanguinaria F. D. Ott and group, as proposed by Cavalier-Smith (1999), are Porphyridium aerugineum Geitler; Rhodella violacea dominant members of marine phytoplankton (Kornmann) Wehrmeyer and Dixoniella grisea (Gei- assemblages, including diatoms, dinoflagellates, tler) J. L. Scott, S. T. Broadwater, B. D. Saunders, haptophytes, and cryptophytes (Falkowski et al. J. P. Thomas et P. W. Gabrielson; and Stylonema alsi- 2004, Falkowski and Knoll 2007, Simon et al. dii (Zanardini) K. M. Drew. In Bayesian inference 2009). The evolutionary history of the chromalveo- and maximum-likelihood (ML) phylogenetic analy- lates is complex, and the composition of the super- ses, chromalveolate GSII sequences formed a weakly group and relationships among lineages are under supported clade that nested among sequences from continued revision (Parfrey et al. 2010). Currently, glaucophytes, red algae, green algae, and plants. Red it is well established that these organisms gained algal GSII sequences formed two distinct clades. their plastid from red-alga-like endosymbiont (Bhatta- The largest clade contained representatives from the charya et al. 2004, Keeling 2004, Archibald 2005), Cyanidiophytina and Rhodophytina and grouped and recent research indicates some lineages may with plants and green algae. The smaller clade have acquired green algal genes at some point in (C. caeruleus, Porphyra yezoensis, and S. alsidii) nested their evolutionary history (Frommolt et al. 2008, within the chromalveolates, although its placement Moustafa et al. 2009). Moustafa et al. (2009) pro- was unresolved. Chromalveolate GSIII sequences posed that the acquisition of genes from these two formed a well-supported clade in Bayesian and ML primary photosynthetic lineages (red and green phylogenies, and mitochondrial transit peptides were algae) may have provided the chromalveolates with identified in many of the sequences. There was the genetic composition to become ecologically suc- strong support for a stramenopile-haptophyte-crypto- cessful and dominate primary productivity in the phyte GSIII clade in which the cryptophyte sequence marine ecosystem. diverged from the deepest node. Overall, the evolu- Nitrogen availability is a key factor regulating pri- tionary history of the GS gene families within the mary productivity in marine ecosystems. Inorganic algae is complex with evidence for the presence of + forms of nitrogen are reduced to NH prior to the orthologous and paralogous sequences, ancient and 4 incorporation into organic compounds and gluta- recent gene duplications, gene losses and replace- mine synthetase (GS: EC 6.3.1.2) is an enzymatic ments, and the potential for both endosymbiotic and link between carbon and nitrogen metabolism as it lateral gene transfers. + catalyzes the ATP-dependent condensation of NH4 Key index words: chromalveolates; chromists; cryp- and glutamate forming glutamine. The nitrogen tophytes; dinoflagellates; glutamine synthetase II; incorporated into glutamine by GS is transferred to 2-oxoglutarate by the activity of glutamine: 2-oxo- glutarate amidotransferase (GOGAT, EC 1.4.7.1 or 1Received 9 November 2010. Accepted 3 November 2011. EC 1.4.1.13) yielding two molecules of glutamate. 2Author for correspondence: e-mail [email protected]. The glutamine and glutamate produced by the 768 GSII AND GSIII IN CHROMALVEOLATES 769 GS:GOGAT cycle are used in a wide variety of essential are expressed in Porphyridium cruentum. A single biosynthetic pathways in the cell. GSII sequence was identified in the genome of Cya- Photosynthetic eukaryotes usually express multi- nidiophyceae, Cyanidioschyzon merolae (Matsuzaki ple GS isoenzymes that function in the cytosol and et al. 2004), and biochemical studies showed that chloroplast. GS isoenzymes are also targeted to the the enzyme functions in the cytosol (Terashita et al. mitochondria in some vascular plants (Taira et al. 2006). GSII sequences were also reported from Gal- 2004), which may be true for other photosynthetic dieria sulphuraria (Cyanidiophyceae) in an EST study eukaryotes (Allen et al. 2006). The GS isoenzymes (Weber et al. 2004), and a GSII cDNA was charac- comprise a protein superfamily that has three dis- terized from Gelidium crinale (Florideophyceae; tinct classes (GSI, GSII, and GSIII), which differ in Freshwater et al. 2002). Phylogenetic analyses sug- molecular size and the number of subunits compris- gested a sister association between the G. crinale ing the holoenzyme (Brown et al. 1994, Eisenberg GSII and that of green algae, but the association et al. 2000). Within streptophytes, the GS isoen- was not well supported (Freshwater et al. 2002). zymes are members of the GSII gene family and Two subphyla, the Cyanidiophytina and Rhodo- appear to have evolved via recent gene duplication, phytina, are currently recognized within Rhodo- with an expansion in the number of genes encoding phyta (Yoon et al. 2006). Phylogenetic analyses of cytosolic-localized isoenzymes (Coruzzi et al. 1989, plastid encoded genes of red algae and chromists Ghoshroy et al. 2010). Multiple GSII isoenzymes are suggest that the secondary endosymbiotic event also expressed in green algae and early diverging involving a red alga occurred after the divergence plants; however, the genes encoding the chloro- of members of Cyanidiophytina from the Rhodophy- plast-targeted isoenzyme appear to have evolved via tina and before the divergence of the current Flor- a horizontal gene transfer (HGT) from eubacteria ideophyceae group from the other lineages in the to green algae early in plant evolution (Ghoshroy Rhodophytina (Yoon et al. 2002, 2004). Thus, mem- et al. 2010). Although, there is biochemical bers of the Rhodophytina, comprising the former evidence of multiple GS isoenzymes in red algae Bangiophyceae, are predicted to be the putative (Casselton et al. 1986), to date, molecular studies donor of secondary plastids in the chromalveolates. have only identified genes encoding cytosolic-local- Characterization of GSII from members of the ized enzymes (Freshwater et al. 2002, Terashita et al. Rhodophytina is needed to further explore the 2006). hypothesis that GSII in chromalveolates evolved by Multiple GS isoenzymes are also expressed in the EGT. chromalveolates. However, in contrast to plants and Another key group for understanding the evolu- green algae, the isoenzymes are members of the tion of the chromalveolates and the molecular evo- GSII and GSIII superfamilies. Within the chromalve- lution of GSII are the cryptophytes. Cryptophytes olates, GSII genes have been reported from stra- are unique in that they retain the nuclear genome menopiles (Armbrust et al. 2004, Robertson and of the red algal endosymbiont as the reduced nucle- Tartar 2006, Bowler et al. 2008, Nedelcu et al. omorph. Several phylogenetic studies using plastid- 2009), haptophytes (Nedelcu et al. 2009), dinofla- encoded genes suggested that cryptophytes were an gellates, and nonphotosynthetic alveolates (Slamovits early diverging clade within the chromists (Yoon and Keeling 2008). Phylogenetic analyses of GSII in et al. 2002, 2004), and single- and multigene analy- diatoms and the nonphotosynthetic oomycetes sug- ses have recovered a sister association between gested that GSII evolved via an endosymbiotic gene haptophytes and cryptophytes (reviewed in Keeling transfer (EGT) from the nuclear genome of the 2009). More recent phylogenetic analyses using mul- photosynthetic endosymbiont to the nuclear gen- tigene data sets have grouped the stramenopiles, ome of the host cell (Robertson and Tartar 2006, alveolates, and rhizaria into a clade that excluded Slamovits and Keeling 2008). If true, this possibility cryptophytes and haptophytes (Yoon et al. 2008, suggests that photosynthesis evolved early in the evo- Burki et al. 2009, Parfrey et al. 2010), and other lution of the stramenopiles (Robertson and Tartar studies using nuclear genes failed to recover the 2006). Phylogenetic analyses showed that GSII from monophyly of the chromalvelotes (Parfrey et al. the nonphotosynthetic