title: Cloning, molecular characterization, and phylogeny of two evolutionary distinct glutamine synthetase isoforms in the green microalga Haematococcus pluvialis (Chlorophyceae).
abstract: Haematococcus pluvialis (Chlorophyta) is a widely used microalga of great economic potential, yet its molecular genetics and evolution are largely unknown. We present new detailed molecular and phylogenetic analysis of two glutamine synthetase (GS) enzymes and genes (gln) under the Astaxanthin-inducing conditions of light- and nitrogen-stress. Structure analysis identified key residues and confirmed two decameric GS2holoenzymes, a cytoplasmic enzyme, termed GS2c, and a plastidic form, termed GS2p, due to chloroplast-transit peptides at its N-terminus. Gene expression analysis showed dissociation of mRNA, protein, and enzyme activity levels for both GS2under different growth conditions, indicating the strong post-transcriptional regulation. Data-mining identified novel and specified published gln genes from Prasinophyceae, Chlorophyta, Trebouxiophyceae, Charophyceae, Bryophyta, Lycopodiophyta, Spermatophyta, and Rhodophyta. Phylogenetic analysis found homologues to the cytosolic GS2cof H. pluvialis in all other photo- and non-photosynthetic Eukaryota. The chloroplastic GS2pwas restricted to Chlorophyta, Bryophyta, some Proteobacteria and Fungii; no homologues were identified in Spermatophyta or other Eukaryota. This indicates two independent prokaryotic donors for these two gln genes in H. pluvialis. Combined phylogenetic analysis of GS, chl-b synthase, elongation factor, and light harvesting complex homologues project a newly refined model of Viridiplantae evolution. Herein, a GS1evolved into the cytosolic GS2cand was passed on to all Eukaryota. Later, the chloroplastic GS2pentered the Archaeplastida lineage via a horizontal gene transfer at the divergence of Chlorophyta and Rhodophyta lineages. GS2ppersisted in Chlorophyta and Bryophyta, but was lost during Spermatophyta evolution. These data suggest the revision of GS classification and nomenclature, and extend our understanding of the photosynthetic Eukaryota evolution.
authors.affiliation: Food and Bioprocess Engineering Group, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen, the Netherlands.
authors.forename: Diana L
authors.initials: DL
authors.lastname: Reinecke
authors.affiliation: Microalgal Biotechnology Lab, Blaustein Institutes of Desert Research, Ben-Gurion University of the Negev, Sede-Boqer Campus, Sede-Boqer, 84990, Israel.
authors.forename: Aliza
authors.initials: A
authors.lastname: Zarka
authors.affiliation: Microalgal Biotechnology Lab, Blaustein Institutes of Desert Research, Ben-Gurion University of the Negev, Sede-Boqer Campus, Sede-Boqer, 84990, Israel.
authors.forename: Stefan
authors.initials: S
authors.lastname: Leu
authors.affiliation: Microalgal Biotechnology Lab, Blaustein Institutes of Desert Research, Ben-Gurion University of the Negev, Sede-Boqer Campus, Sede-Boqer, 84990, Israel.
authors.forename: Sammy
authors.initials: S
authors.lastname: Boussiba
mesh_terms: D020418:Algal Proteins / Q000235:genetics*; D000595:Amino Acid Sequence; D000460:Chlorophyta / Q000145:classification* / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D058086:Microalgae / Q000145:classification / Q000201:enzymology / Q000235:genetics; D010802:Phylogeny; D016415:Sequence Alignment
chemical_list: D020418:Algal Proteins; D005974:Glutamate-Ammonia Ligase
keywords: Archaeplastida; Astaxanthin; Charophyta; Lycopodiophyta; alga biotechnology; glutamine synthetase; green plant evolution; horizontal gene transfer; nitrogen uptake; transcript regulation