CLONING, MOLECULAR CHARACTERIZATION, AND PHYLOGENY OF TWO EVOLUTIONARY DISTINCT GLUTAMINE SYNTHETASE ISOFORMS IN THE GREEN MICROALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE)

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 GS(2) holoenzymes, a cytoplasmic enzyme, termed GS(2c), and a plastidic form, termed GS(2p), due to chloroplast-transit peptides at its N-terminus. Gene expression analysis showed dissociation of mRNA, protein, and enzyme activity levels for both GS(2) under 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 GS(2c) of H. pluvialis in all other photoand non-photosynthetic Eukaryota. The chloroplastic GS(2p) was 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 GS(1) evolved into the cytosolic GS(2c) and was passed on to all Eukaryota. Later, the chloroplastic GS(2p) entered the Archaeplastida lineage via a horizontal gene transfer at the divergence of Chlorophyta and Rhodophyta lineages. GS(2p) persisted 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.