Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution
© Ghoshroy et al; licensee BioMed Central Ltd. 2010
Received: 23 December 2009
Accepted: 25 June 2010
Published: 25 June 2010
Glutamine synthetase (GS) is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII) are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria) to the Chloroplastida.
GSII sequences were isolated from four species of green algae (Trebouxiophyceae), and additional green algal (Chlorophyceae and Prasinophytae) and streptophyte (Charales, Desmidiales, Bryophyta, Marchantiophyta, Lycopodiophyta and Tracheophyta) sequences were obtained from public databases. In Bayesian and maximum likelihood analyses, eubacterial (GSIIB) and eukaryotic (GSIIE) GSII sequences formed distinct clades. Both GSIIB and GSIIE were found in chlorophytes and early-diverging streptophytes. The GSIIB enzymes from these groups formed a well-supported sister clade with the γ-Proteobacteria, providing evidence that GSIIB in the Chloroplastida arose by horizontal gene transfer (HGT). Bayesian relaxed molecular clock analyses suggest that GSIIB and GSIIE coexisted for an extended period of time but it is unclear whether the proposed HGT happened prior to or after the divergence of the primary endosymbiotic lineages (the Archaeplastida). However, GSIIB genes have not been identified in glaucophytes or red algae, favoring the hypothesis that GSIIB was gained after the divergence of the primary endosymbiotic lineages. Duplicate copies of the GSIIB gene were present in Chlamydomonas reinhardtii, Volvox carteri f. nagariensis, and Physcomitrella patens. Both GSIIB proteins in C. reinhardtii and V. carteri f. nagariensis had N-terminal transit sequences, indicating they are targeted to the chloroplast or mitochondrion. In contrast, GSIIB proteins of P. patens lacked transit sequences, suggesting a cytosolic function. GSIIB sequences were absent in vascular plants where the duplication of GSIIE replaced the function of GSIIB.
Phylogenetic evidence suggests GSIIB in Chloroplastida evolved by HGT, possibly after the divergence of the primary endosymbiotic lineages. Thus while multiple GS isoenzymes are common among members of the Chloroplastida, the isoenzymes may have evolved via different evolutionary processes. The acquisition of essential enzymes by HGT may provide rapid changes in biochemical capacity and therefore be favored by natural selection.
Glutamine synthetase (GS: E.C. 184.108.40.206) catalyzes the ATP-dependent formation of Gln from Glu and NH4 + and is considered one of the oldest functioning enzymes [1, 2]. The GS gene superfamily includes three distinct classes, GSI, GSII and GSIII, each differing in molecular size and number of subunits in the holoenzyme [3, 4]. The distribution of the three classes is variable within the three domains of life and instances of multiple GS isoenzymes from different families functioning in the same organism are not uncommon in both eubacteria and eukaryotes [3, 5–8]. These observations suggest the gene families arose early and prior to the divergence of the prokaryotes and eukaryotes [9–11].
The evolutionary history of the GS superfamily is complicated and gene transfer events are one among many forces that have shaped the history of these genes. Among prokaryotes, there is phylogenetic evidence of horizontal gene transfer (HGT) of GSI genes between members of the Archaea and Eubacteria . Evidence for secondary endosymbiotic gene transfer of GSII from the red algal endosymbiont to the nucleus of the heterokont host has also been presented . Members of the GSII gene family are found in both eubacterial and eukaryotic lineages. The identification of GSII genes in the plant symbiont Bradyrhizobium japonicum lead Carlson and Chelm  to hypothesize that the gene evolved via HGT from vascular plants to bacteria. However, this hypothesis was not supported by subsequent phylogenetic analyses [11, 13], which established distinct eukaryotic (GSIIE) and eubacterial (GSIIB) clades.
The supergroup Archaeplastida , consisting of Glaucophyta, Rhodophyceae and Chloroplastida, harbors members of GSII gene family that are well characterized in vascular plants but not in other lineages within the supergroup. In general, vascular plants express multiple GS isoenzymes that are localized to either cytosol or chloroplast. The isoenzymes are nuclear encoded, and in most angiosperms a single nuclear gene encodes the chloroplast isoenzyme, while a small nuclear gene family encodes multiple cytosolic isoenzymes that are expressed in tissue-specific and developmentally-regulated patterns [15–18]. Previous phylogenetic analyses of chloroplast and cytosolic isoenzymes support the hypothesis that the isoenzymes in angiosperms evolved via a gene duplication event that preceded the divergence of monocots and dicots [19, 20].
Biochemical studies of green algae provided the first evidence that, as observed in vascular plants, multiple GSII isoenzymes are expressed and localized to the cytosol and chloroplasts within these organisms [21, 22]. Phylogenetic analyses incorporating the two GSII isoforms characterized in Chlamydomonas reinhardtii  uncovered an unusual disparity between the two enzymes . The cytosolic GSII sequence clustered with the vascular plants while the plastid sequence branched more basally and appeared to associate with the eubacterial sequences.
Here we examined the evolutionary relationship of the GSII gene family and use increased taxonomic sampling in Chloroplastida to determine if the basally branching, eubacterial-like GSIIB was broadly distributed. GSII sequences were obtained from four members of the Trebouxiophyceae (Chlorophyta) by PCR amplification using degenerate and gene specific primers. Additional GSII sequences for members of the green algae (Chlorophyta and Prasinophytae) and streptophytes (Mesostigma, Charales, Desmidiales, Bryophyta, Marchantiophyta, Lycopodiophyta and Tracheophyta) were obtained from publicly available databases, including genome and EST projects. We also increased taxonomic sampling within Eubacteria (to date, GSII genes have not been reported from Archaea). GSIIE and GSIIB sequences were identified in members of the green algae and early-diverging streptophytes. Phylogenetic analyses provide support for the hypothesis that GSIIB was gained in the Chloroplastida from the Eubacteria via a HGT event after the divergence of primary photosynthetic groups.
Results and Discussion
Amplification of GSII genes
Summary of the GSII sequences characterized in the present study
% GC content
GSII E Sequences
Pseudochlorella sp. CCAP211/1A (2)
Chlorella luteoviridis UTEX 28
Prototheca zopfii ATCC16527
GSII B Sequences
Pseudochlorella sp. CCAP211/1A (1)
Eukaryotic GSII phylogeny
Evidence for the HGT of GSIIB
GSIIB sequences are not broadly represented among eubacteria but were identified in members of the Bacteriodetes/Flavobacteria/Cytophaga, Planctomycetes, Verrucomicrobia, Actinobacteria, and the α- and γ-Proteobacteria (Figure 3; Additional files 1 and 2). The Chloroplastida GSIIB was sister to γ-Proteobacteria with strong (Bayesian posterior probability = 1.0) to moderate support (likelihood bootstrap support = 70%). The γ-Proteobacteria + Chloroplastida GSIIB clade was sister to the Actinobacteria, but this association was not strongly supported. The α-Proteobacteria GSIIB sequences were not closely related to the γ-Proteobacteria + Chloroplastida GSIIB clade, which makes the possibility of GSIIB gain via mitochondrial endosymbiosis unlikely. The α-Proteobacteria GSIIB were nested within the Verrucomicrobia and thus, we cannot exclude the possibility of an HGT event within the α-Proteobacteria lineage that obscures the mitochondrial origin of the GSIIB gene in the Chloroplastida. However, the lack of detection of GSIIB in genomes of other eukaryotic lineages reduces the likelihood of a mitochondrial origin. In addition, EST and genome analyses of other photosynthetic eukaryotes (Glaucophyta, Rhodophyceae and Chromalveolates) and extant cyanobacteria [26, 27], have not uncovered GSIIB sequences, reducing the possibility that GSIIB was acquired via plastid endosymbiosis. Thus, we propose that GSIIB in the Chloroplastida arose via a HGT from γ-Proteobacteria early in plant evolution.
GSIIB sequences are not broadly distributed among eubacterial lineages and to date, within γ-Proteobacteria, only the genera represented in our analyses have annotated GSIIB sequences deposited in GenBank. Assuming the GS superfamily evolved prior to the divergence of the three domains of life [9–11], the distribution of GSIIB sequences suggests the gene has been lost in several lineages of Eubacteria and the Archaea. The analysis of GSIIB may become more robust as additional eubacterial GSIIB become available through genome sequencing projects. However, gene loss may make the identification of the true donor of GSIIB to the Chloroplastida difficult.
An alternative explanation for the limited distribution of GSIIB among the eubacteria is that the gene was transferred to the eubacteria from an eukaryotic donor. The possibility of an HGT from Chloroplastida to the γ-Proteobacteria is not supported by our phylogenetic analyses as it implies that the eubacterial sequences would nest within the GSIIE clade; which has not been observed in our phylogenetic analyses. Eukaryote to eubacterial HGT might be supported if GSIIB were found in diverse lineages of eukaryotes. Further investigation of GSII diversity in the eukaryotic lineages not represented in our study (e.g., Rhizaria, Excavata and Amoebozoa) will contribute to our understanding of the distribution and evolution of GSIIB. Given the data at hand, however, the hypothesis that GSIIB arose in the Chloroplastida via HGT remains the most parsimonious.
Estimating the timing of the HGT
The distribution of GSIIB within Chloroplastida covers the major lineages of Chlorophyta (Chlorophyceae, Trebouxiophyceae, and Prasinophyceae; Additional file 1). In addition, a partial GSIIB sequence was identified in a member of the Ulvophyceae (Acetabularia acetabulum; (Additional files 2 and 4). Within Streptophyta, GSIIB genes are present in Mesostigmatophyceae (Mesostigma viride; Additional files 2 and 4), Zygnemophyceae (Desmidiales; Closterium peracerosum-strigosum-littorale complex), Marchantiophyta (liverworts; Marchantia polymorpha) and Bryophyta (mosses; Physcomitrella patens). GSIIB is absent from the single Lycopodiophyta genome (Selaginella moellendorffii) and from all seed plants. Hence, we propose that GSIIB was lost in the plant lineage after the colonization of land by early plants, marked by the divergence of bryophytes and lycopodiophytes, which is one of the oldest vascular plant lineages .
Functional localization and GSIIBgene duplication
The Chloroplastida lineages that contain the GSIIB gene also have a GSIIE counterpart, which attaches to a basal node within the photosynthetic eukaryotes (Figure 1). Both the GSIIB and GSIIE genes are nuclear encoded and thus we identified the cellular location of each of the gene products based on the presence (organelle-localized) or absence (cytosol-localized) of N-terminal transit peptides using TargetP ver. 1.1 (, see Additional file 5). None of the early-diverging Chloroplastida GSIIE enzymes contained transit peptides. In contrast, chloroplast transit sequences were identified in the GSIIB protein sequences from Chlorella sp. NC64A, C. vulgaris and the streptophyte, Closterium peracerosum-strigosum-littorale (Zygnemophyceae) but not in the moss (P. patens) or liverwort (M. polymorpha). Mitochondrial-targeting transit peptides were predicted in GSIIB sequences from C. reinhardtii, Volvox carteri f. nagariensis and Scenedesmus obliquus (see Additional file 5). Previous work indicated that chloroplast transit sequences from C. reinhardtii shared features with both mitochondrial and higher plant chloroplast pre-sequences  and thus the prediction of a mitochondrial location of GSIIB may not reflect its true functional localization. Alternatively, GSIIB may be targeted to both the mitochondria and chloroplast, similar to what is observed for GSIIE in leaves of some vascular plants [35, 36]. While experimental evidence is required to confirm the cellular localization of the GSIIB, it appears that the GSIIB enzymes function in either the chloroplast or mitochondrion in the chlorophytes and early-diverging streptophytes (Closterium sp.) and that GSIIE functions in the cytosol.
The GSIIB gene is duplicated in C. reinhardtii, V. carteri f nagariensis and P. patens. The duplicated copies of GSIIB in C. reinhardtii and V. carteri f nagariensis were nearly identical (90% and 95% identical, respectively) and present in the genome in a head-to-head orientation. Similarly, the GSIIB genes in P. patens were 98% identical but do not appear to be in close genomic proximity. Within our phylogenetic analyses (Figure 3), the duplicated GSIIB of C. reinhardtii, V. carteri f. nagariensis and P. patens each formed separate clades, suggesting the genes evolved by independent duplication events. Alternatively, the GSIIB genes in C. reinhardtii and V. carteri may have evolved via an early duplication within the Chlamydomonadales with subsequent gene conversion following the divergence of these lineages. The GSIIB are differentially expressed in C. reinhardtii suggesting the need for maintenance of both the copies in the organism .
GSIIBloss and replacement of function
In contrast to the expression of GSIIE and GSIIB genes in the early-diverging Chloroplastida, the chloroplast- and cytosolic-localized GSII enzymes in angiosperms are both members of the GSIIE family and form two distinct clades in our phylogenetic analyses (Figures 1 and 2). As predicted in earlier studies , the genes encoding these enzymes arose via a recent gene duplication event with further expansion in the number of genes encoding cytosolic isoenzymes in several plant lineages (Figure 2, [38, 39]). Since GSIIB is absent from vascular plants, it appears that the chloroplast function of GSIIB has been replaced by a gene duplication event in higher plants allowing for subsequent loss of the gene from this lineage. There is also an expansion of the GSIIE gene family in gymnosperms (Figure 2), but the enzymes are all localized to the cytosol and the plastid targeted isoform appears to have been lost from this group. The expansion of the GSIIE gene family coincides with the development of vascularization of land plants and maybe correlated with the partitioning of nitrogen assimilation between below and above ground tissue (see Additional file 3).
We have provided evidence of an ancient HGT event involving the gene for an essential enzyme, GSII. GSII has been well characterized at the molecular level in angiosperms but has been largely overlooked in the early-diverging plant lineages, which were addressed in the present study. Although recent comparative genomic analyses failed to identify bacterial genes in Chlamydomonas reinhardtii , our discovery of a eubacterial-like GSII in the chlorophytes and early-diverging streptophytes suggests that further exploration within these lineages is merited. The branching pattern within the monophyletic assemblage of the chlorophytes and early-diverging streptophytes is similar to other molecular and organismal phylogenies, suggesting the occurrence of a single HGT event. As a result, GSIIB may be useful in resolving taxonomic associations within and among green algal and early-diverging streptophyte lineages.
Several genes of bacterial origin have been identified in Dictyostelium discoideum and are thought to be advantageous to organisms living in soil . More recently, Richards et al.  identified five genes in plants that appear to be of fungal origin and argue that two may have been advantageous for organisms colonizing a terrestrial environment. We propose that the acquisition of enzymes by HGT results in a more rapid change in enzymatic capacity or kinetic diversity than evolution of isoenzymes by gene duplication and subsequent specialization. Biochemical studies have suggested that GSIIB has a lower affinity for NH4 + and Glu than GSIIE , characteristics that would be advantageous for enzymes assimilating higher concentrations of NH4 + from environmental sources, NO3 - assimilation, or increased rates of photorespiration. Increased taxon sampling and an enlarged fossil age constraint dataset will allow for a more detailed examination of the timing of GSII gains and losses over geological history and coupled with major transitions in plant evolution.
Algal cultures and sequencing
Primers used for amplification of GSII genes from green algae
Green UNI 1-F
GALG GS F
5' - TGC CCA TCC CCA CCA ACA C - 3'
GALG GS R
5' - TCT CGT GCT TGC CCG TCA GG - 3'
5' - CGG CWT CGA GCA GGA GTA CAC - 3'
5' - CCG AYC TGG WAC TCC CAC TGG - 3'
Nested PCR amplification was used to obtain GSIIB sequences with first round done with cDNA and primer concentrations of 0.4 μM (MossGS2-1F [forward] and MossGS2-2R [reverse]). The amplicon was used for a second round of amplification with primers Green UNI 1-F (forward) and cpGSII(QGPFY)-R (reverse) and yielded a DNA fragment of 330 bp. Amplified sequences were cleaned and sequenced either commercially (MWG Biotech, Charlotte, NC and Macrogen, Seoul, South Korea) or at Clark University using an automated DNA sequencer (ABI 3130), with ABI Prism Terminator Big Dye ver 3.1 (Applied Biosystems, Carlsbad, CA). Some PCR products were cloned into TOPO vectors following the manufacturer's protocol (TOPO TA Cloning Kit for Sequencing, Invitrogen, Carlsbad, CA) prior to sequencing. Rapid Amplification of cDNA Ends (RACE) methods were used to obtain the entire open reading frame for GSIIE sequences from Pseudochlorella sp. CCAP211/1A, C. luteoviridis, A. protothecoides and P. zopfii and partial GSIIB sequence from Pseudochlorella sp. CCAP211/1A. 3' RACE reactions used a combination of gene specific primers and a portion of the oligo-d (T) primer (GCGGCCGCTCTAGACTAGT) used for cDNA synthesis. 5' RACE reactions were performed using 5' RACE System version 2.0 from Invitrogen (Invitrogen) and SMART™ RACE cDNA Amplification Kit (Clontech Laboratories Inc., Mountain View, CA) following manufacturers' recommendations. Contigs were assembled using CodonCode Aligner (CodonCode Corporation, Deadham, MA). All sequences were translated into amino acids in silico.
GSII sequences were retrieved from public databases as well as genome and EST projects using the GSII sequence from the diatom Skeletonema costatum (AAC77446) as query, or glutamine synthetase as a keyword. Subsequent queries with eubacterial GSII sequences did not retrieve any additional sequences. Complete information on taxa, database sources and accession numbers is provided in Additional file 1. The initial alignment of amino acid sequences was done with the web based program CLUSTAL W, using default parameters , followed by manual adjustment using BioEdit Sequence Alignment Editor  and MacClade 4.08 . The N- and C terminal ends of the proteins along with highly variable regions within the alignments were excluded in the phylogenetic analyses.
The final GSII alignment consisted of 196 taxa and 333 characters for Bayesian analysis. Trees were inferred by calculating Bayesian posterior probabilities using MrBayes 3.1.2 [48, 49]. Two parallel runs, each with four chains (three heated and one cold) were run for 106 generations. The evolutionary models implemented in MrBayes3.1.2 were explored using the mixed amino acid model. Rate variation across sites was approximated using a gamma distribution with proportion of invariable sites estimated from the data. Trees were sampled every 100 generations. Likelihood tree scores of two independent runs were plotted to estimate the point of convergence to a stable likelihood, and to determine the trees to be excluded via "burnin." Bayesian posterior probabilities of the branches were calculated from trees from both the runs, totaling 20,002 trees. Trees remaining (10,000) after a burnin of 5001 for each run were used to compute a 50% majority-rule consensus.
Maximum likelihood (ML) based inference of the phylogenetic trees was done using the software RAxML 7.0.4 [50, 51]. The analysis used a random starting tree and the rapid hill-climbing algorithm (i.e., option -f d in RAxML) and the WAG model of amino acid substitution were used. A random seed number was used to turn on rapid bootstrapping (-x) and 1000 bootstrap trees were generated by invoking -# 1000 and - x options in RAxML. A majority rule consensus tree was created in PAUP* 4.0 b . The phylogenetic trees in figures 1, 2 & 3 are the 50% majority rule consensus trees from the Bayesian analyses on which the RAxML bootstrap values have been indicated. The eubacterial GSIIB sequences were used as the monophyletic outgroup in the graphical representation of the phylogenies.
Prediction of functional localization of GSIIB and GSIIEprotein sequences in early-diverging Chloroplastida
Estimation of divergence times
We estimated the divergence times using Bayesian approach implemented in BEAST 1.4.8 . We did an un-calibrated and calibrated run. A relaxed molecular clock model of uncorrelated log normal distribution was used. For the un-calibrated analysis, a starting tree generated by RAxML 7.0.4  was used as the input tree with the GS amino acid sequence alignment. For the calibrated analysis we set uniform priors on tmrca parameter. Fossil dates were used as minimum dates and were, as follows, Ascomycota, 400 MYA , Bilateria, 550 MYA  and streptophytes 475 MYA . Secondary age constraints based on published estimates of divergence times were not used. We used the following models, WAG + Inv + Gamma with priors, birth death speciation on the tree. Markov Chain Monte Carlo was set to default 10 million with sampling at every 1000 generation, resulting in 10,000 trees. Convergence was assessed in Tracer v 1.4  and the first three million samples were excluded as burnin. A maximum clade credibility tree was generated by analyzing the BEAST tree file in TreeAnnotator 1.4.6 . This program determined the 95% highest posterior densities and estimated the node heights as mean heights.
Horizontal gene transfer
Bayesian posterior probability
We thank Jenna Nguyen and Jacqueline Mitchell for their assistance with this project and David Hibbett for helpful discussions regarding the phylogenetic analyses. We also thank two anonymous reviewers and the editor for their constructive comments and recommendations. This research was supported by an NSF CAREER Award (IBN 0238426) to DLR.
- Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y: Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci USA. 1993, 90: 3009-3013. 10.1073/pnas.90.7.3009.PubMed CentralView ArticlePubMed
- Pesole G, Bozzetti MP, Lanave C, Preparata G, Saccone C: Glutamine synthetase gene evolution: A good molecular clock. Proc Natl Acad Sci USA. 1991, 88: 522-526. 10.1073/pnas.88.2.522.PubMed CentralView ArticlePubMed
- Brown JR, Masuchi Y, Robb FT, Doolittle WF: Evolutionary relationships of bacterial and archaeal glutamine synthetase genes. J Mol Evol. 1994, 38: 566-576. 10.1007/BF00175876.View ArticlePubMed
- Eisenberg D, Gill HS, Pfluegl GMU, Rotstein SH: Review: Structure-function relationships of glutamine synthetases. Biochim Biophys Acta. 2000, 1477: 122-145.View ArticlePubMed
- Garcia-Dominguez M, Reyes JC, Florencio FJ: Purification and characterization of a new type of glutamine synthetase from cyanobacteria. Eur J Biochemistry. 1997, 244: 258-264. 10.1111/j.1432-1033.1997.00258.x.View Article
- Reyes JC, Florencio FJ: A new type of glutamine synthetase in cyanobacteria: The protein encoded by the glnN gene supports nitrogen assimilation in Synechocystis sp. strain PCC 6803. J Bacter. 1994, 176: 1260-1267.
- Robertson DL, Alberte RS: Isolation and characterization of glutamine synthetase from the marine diatom Skeletonema costatum. Plant Physiol. 1996, 111: 1169-1175. 10.1104/pp.111.4.1169.PubMed CentralView ArticlePubMed
- Robertson DL, Smith GJ, Alberte RS: Glutamine synthetase in marine algae: new surprises from an old enzyme. J Phycol. 2001, 37: 793-795. 10.1046/j.1529-8817.2001.01057.x.View Article
- Brown JR, Doolittle WF: Archaea and the prokaryote-to-eukaryote transition. Microbiol Mol Biol Rev. 1997, 61: 456-502.PubMed CentralPubMed
- Mathis R, Gamas P, Meyer Y, Cullimore JV: The presence of GSI-like genes in higher plants: Support for the paralogous evolution of GSI and GSII. J Mol Evol. 2000, 50: 116-122.PubMed
- Robertson DL, Tartar A: Evolution of glutamine synthetase in heterokonts: evidence for endosymbiotic gene transfer and the early evolution of photosynthesis. Mol Biol Evol. 2006, 23: 1048-1055. 10.1093/molbev/msj110.View ArticlePubMed
- Carlson TA, Chelm BK: Apparent eucaryotic origin of glutamine synthetase II from the bacterium Bradyrhizobium japonicum. Nature. 1986, 568-570. 10.1038/322568a0.
- Shatters RG, Kahn ML: Glutamine synthetase II in Rhizobium: reexamination of the proposed horizontal transfer of DNA from eukaryotes to prokaryotes. J Mol Evol. 1989, 29: 422-428. 10.1007/BF02602912.View ArticlePubMed
- Adl SM, Simpson AGB, Farmer MA, Andersen RA, Anderson OR, Barta JR, Bowser SS, Brugerolle G, Fensome RA, Fredericq S, James TY, Karpov S, Kugrens P, Krug J, Lane CE, Lewis LA, Lodge J, Lynn DH, Mann DG, Mccourt RM, Mendoza L, Moestrup Ø, Mozley-Standridge SE, Nerad TA, Shearer CA, Smirnov AV, Spiegel FW, Taylor MFJR: The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol. 2005, 5: 399-451. 10.1111/j.1550-7408.2005.00053.x.View Article
- Coruzzi GM: Primary N-assimilation into Amino Acids in Arabidopsis. The Arabidopsis Book. Edited by: Somerville C, Meyerowitz E. 2003, Rockville, MD: American Society of Plant Biologists, 1-17. 10.1199/tab.0010.
- Lam H-M, Coschigano KT, Oliveira IC, Melo-Oliveira R, Coruzzi GM: The molecular-genetics of nitrogen assimilation into amino acids in higher plants. Annu Rev Plant Physiol Mol Biol. 1996, 47: 569-593. 10.1146/annurev.arplant.47.1.569.View Article
- Sechley KA, Yamaya T, Oaks A: Compartmentation of nitrogen assimilation in higher plants. Int Rev Cytol. 1992, 134: 85-163. 10.1016/S0074-7696(08)62028-8.View Article
- Woodall J, Boxall JG, Forde BG, Pearson J: Changing perspectives in plant nitrogen metabolism: the central role of glutamine synthetase. Science Progress. 1996, 79: 1-26.
- Coruzzi GM, Edwards JW, Tingey SV, Tsai F-Y, Walker EL: Glutamine synthetase: molecular evolution of an eclectic multi-gene family. The Molecular Basis of Plant Development. Proceedings of an E.I. duPont Nermous-UCLA Symposium. Edited by: Goldberg R. 1989, New York: Alan R. Liss, Inc, 223-232.
- Robertson DL, Smith GJ, Alberte RS: Characterization of a cDNA encoding glutamine synthetase from the marine diatom Skeletonema costatum (Bacillariophyceae). J Phycol. 1999, 35: 786-797. 10.1046/j.1529-8817.1999.3540786.x.View Article
- Ahmad I, Hellebust JA: Nitrogen metabolism of the marine microalga Chlorella autotrophica. Plant Physiol. 1984, 76: 658-663. 10.1104/pp.76.3.658.PubMed CentralView ArticlePubMed
- Casselton PJ, Chandler G, Shah N, Stewart GR, Sumar N: Glutamine synthetase isoforms in algae. New Phytol. 1986, 102: 261-270. 10.1111/j.1469-8137.1986.tb00580.x.View Article
- Chen Q, Silflow CD: Isolation and characterization of glutamine synthetase genes in Chlamydomonas reinhardtii. Plant Physiol. 1996, 112: 987-996. 10.1104/pp.112.3.987.PubMed CentralView ArticlePubMed
- Suarez MF, Avila C, Gallardo F, Canton FR, Garcia-Gutierrez A, Claros MG, Canovas FM: Molecular and enzymatic analysis of ammonium assimilation in woody plants. J Exp Bot. 2002, 53: 891-904. 10.1093/jexbot/53.370.891.View ArticlePubMed
- Bowman JL, Floyd SK, Sakakibara K: Green genes - comparative genomics of the green branch of life. Cell. 2007, 129: 229-234. 10.1016/j.cell.2007.04.004.View ArticlePubMed
- Dufresne A, Salanoubat M, Partensky Fdr, Artiguenave Fo, Axmann IM, Barbe Vr, Duprat S, Galperin MY, Koonin EV, Le Gall F, Makarova KS, Ostrowski M, Oztas S, Robert C, Rogozin IB, Scanlan DJ, de Marsac NT, Weissenbach J, Wincker P, Wolf YI, Hess WR: Genome sequence of the cyanobacterium Prochlorococcus marinus SS120, a nearly minimal oxyphototrophic genome. Proc Natl Acad Sci USA. 2003, 100: 10020-10025. 10.1073/pnas.1733211100.PubMed CentralView ArticlePubMed
- Palenik B, Brahamsha B, Larimer FW, Land M, Hauser L, Chain P, Lamerdin J, Regala W, Allen EE, McCarren J, Paulsen I, Dufresne A, Partensky F, Webb EA, Waterbury J: The genome of a motile marine Synechococcus. Nature. 2003, 424: 1037-1042. 10.1038/nature01943.View ArticlePubMed
- Drummond AJ, Rambaut A: BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol. 2007, 7: 214-10.1186/1471-2148-7-214.PubMed CentralView ArticlePubMed
- Matsuzaki M, Misumi O, Shin-i T, Maruyama S, Takahara M, Miyagishima S-y, Mori T, Nishida K, Yagisawa F, Nishida K, Yoshida Y, Nishimura Y, Nakao S, Kobayashi T, Momoyama Y, Higashiyama T, Minoda A, Sano M, Nomoto H, Oishi K, Hayashi H, Ohta F, Nishizaka S, Haga S, Miura S, Morishita T, Kabeya Y, Terasawa K, Suzuki Y, Ishii Y, et al: Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature. 2004, 428: 653-657. 10.1038/nature02398.View ArticlePubMed
- Barbier G, Oesterhelt C, Larson MD, Halgren RG, Wilkerson C, Garavito RM, Benning C, Weber APM: Comparative genomics of two closely related unicellular thermo-acidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of Galdieria sulphuraria and significant differences in carbohydrate metabolism of both algae. Plant Physiol. 2005, 137: 460-474. 10.1104/pp.104.051169.PubMed CentralView ArticlePubMed
- Weber A, Oesterhelt C, Gross W, Bräutigam A, Imboden L, Krassovskaya I, Linka N, Truchina J, Schneidereit J, Voll H, Voll L, Zimmermann M, Jamai A, Riekhof W, Yu B, Garavito R, Benning C: EST-analysis of the thermo-acidophilic red microalga Galdieria sulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts. Plant Mol Biol. 2004, 55: 17-32. 10.1007/s11103-004-0376-y.View ArticlePubMed
- Weng J-K, Tanurdzic M, Chapple C: Functional analysis and comparative genomics of expressed sequence tags from the lycophyte Selaginella moellendorffii. BMC Genomics. 2005, 6: 85-10.1186/1471-2164-6-85.PubMed CentralView ArticlePubMed
- Emanuelsson O, Nielsen H, Brunak S, von Heijne G: Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol. 2000, 300: 1005-1016. 10.1006/jmbi.2000.3903.View ArticlePubMed
- Franzén LG, Rochaix JD, von Heijne G: Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences. FEBS Lett. 1990, 260: 165-168. 10.1016/0014-5793(90)80094-Y.View ArticlePubMed
- Taira M, Valtersson U, Burkhardt B, Ludwig RA: Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts. Plant Cell. 2004, 16: 2048-2058. 10.1105/tpc.104.022046.PubMed CentralView ArticlePubMed
- Linka M, Weber APM: Shuffling ammonia between mitochondria and plastids during photorespiration. Trends Plant Sci. 2005, 10: 461-465. 10.1016/j.tplants.2005.08.002.View ArticlePubMed
- Vallon O, Spalding M: Amino Acid Metabolism. The Chlamydomonas Source Book. Edited by: Stern D, Harris EH. 2008, Boston: Elsevier, 2: 115-150. 2
- Doyle JJ, Doyle JL, Harbison C: Chloroplast-expressed glutamine synthetase in Glycine and related Leguminosae: Phylogeny, gene duplication, and ancient polyploidy. Systematic Botany. 2003, 28: 567-577.
- Walker EL, Weeden NF, Taylor CB, Green P, Coruzzi GM: Molecular evolution of duplicate copies of genes encoding cytosolic glutamine synthetase in Pisum sativum. Plant Mol Biol. 1995, 29: 1111-1125. 10.1007/BF00020456.View ArticlePubMed
- Keeling PJ, Palmer JD: Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet. 2008, 9: 605-618. 10.1038/nrg2386.View ArticlePubMed
- Richards TA, Soanes DM, Foster PG, Leonard G, Thornton CR, Talbot NJ: Phylogenomic analysis demonstrates a pattern of rare and ancient horizontal gene transfer between plants and fungi. Plant Cell. 2009, 21: 1897-1911. 10.1105/tpc.109.065805.PubMed CentralView ArticlePubMed
- Florencio FJ, Vega JM: Separation, purification, and characterization of two isoforms of glutamine synthetase from Chlamydomonas reinhardii. Z Naturforsch. 1983, 38c: 531-538.
- Coyer JA, Robertson DL, Alberte RS: Genetic variability within a population and between diploid/haploid tissue of Macrocystis pyrifera (Phaeophyceae). J Phycol. 1994, 30: 545-542. 10.1111/j.0022-3646.1994.00545.x.View Article
- Brown KL, Twing KI, Robertson DL: Unraveling the regulation of nitrogen assimilation in the marine diatom Thalassiosira pseudonana (BACILLARIOPHYCEAE): Diurnal variations in transcript levels for five genes involved in nitrogen assimilation. J Phycol. 2009, 45: 413-426. 10.1111/j.1529-8817.2009.00648.x.View Article
- Thompson JD, Higgins DG, Gibson TJ: Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acid Res. 1994, 22: 4673-4680. 10.1093/nar/22.22.4673.PubMed CentralView ArticlePubMed
- Hall TA: BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999, 41: 95-98.
- Maddison WP, Maddison DR: MacClade 4: Analysis of phylogeny and character evolution. 2000, Sunderland, MA: Sinauer Associates,
- Huelsenbeck J, Ronquist F: MRBAYES: Bayesian inference of phylogeny. Bioinformatics. 2001, 17: 754-755. 10.1093/bioinformatics/17.8.754.View ArticlePubMed
- Ronquist F, Huelsenbeck JP: MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003, 19: 1572-1574. 10.1093/bioinformatics/btg180.View ArticlePubMed
- Stamatakis A: RAxML-VI-HPC: Maximum Likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006, 22: 2688-2690. 10.1093/bioinformatics/btl446.View ArticlePubMed
- Stamatakis A, Hoover P, Rougemont J: A rapid bootstrap algorithm for the RAxML web servers. Syst Biol. 2008, 57: 758-771. 10.1080/10635150802429642.View ArticlePubMed
- Swofford DL: PAUP: Phylogenetic analysis using parsimony, beta version 4.0b1. 1998, Sinauer Associates, Inc., Sunderland
- Emanuelsson O, Nielsen H, von Heijne G: ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Prot Sci. 1999, 8: 978-984. 10.1110/ps.8.5.978.View Article
- Taylor TN, Hass H, Herp H: The oldest fossil ascomycetes. Nature. 1999, 399: 648-10.1038/21349.View ArticlePubMed
- Valentine JW: The macroevolution of phyla. Origin and Early Evolution of the Metazoa. Edited by: Lipps JH, Signor PW. 1992, New York: Plenum Press, 525-553.View Article
- Gray J: Major Paleozoic land plant evolutionary bio-events. Palaeogeogr Palaeoclimatol Palaeoecol. 1993, 104: 153-169. 10.1016/0031-0182(93)90127-5.View Article
- Rambaut A, Drummond A: Tracer v1.4. 2007, [http://beast.bio.ed.ac.uk/Tracer]
- Rambaut A, Drummond AJ: TreeAnnotator 1.4.5. 2006, [http://beast.bio.ed.ac.uk/TreeAnnotator]
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.