Abstract
BackgroundPlants synthesize glutamate from ammonium by the combined activity of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) through the glutamate synthase cycle. In plants, there are two forms of glutamate synthases that differ in their electron donors, NADH-GOGAT (EC 1.4.1.14) and Fd-GOGAT (EC 1.4.7.1), which have differential roles either in primary ammonia assimilation or in the reassimilation of ammonium from different catabolic processes. Glutamate synthases are complex iron-sulfur flavoproteins containing functional domains involved in the control and coordination of their catalytic activities in annual plants. In conifers, partial cDNA sequences for GOGATs have been isolated and used for gene expression studies. However, knowledge of the gene structure and of phylogenetic relationships with other plant enzymes is quite scant.ResultsTechnological advances in conifer megagenomes sequencing have made it possible to obtain full-length cDNA sequences encoding Fd- and NADH-GOGAT from maritime pine, as well as BAC clones containing sequences for NADH-GOGAT and Fd-GOGAT genes. In the current study, we studied the genomic organization of pine GOGAT genes, the size of their exons/introns, copy numbers in the pine genome and relationships with other plant genes. Phylogenetic analysis was performed, and the degree of preservation and dissimilarity of key domains for the catalytic activities of these enzymes in different taxa were determined.ConclusionsFd- and NADH-GOGAT are encoded by single-copy genes in the maritime pine genome. The Fd-GOGAT gene is extremely large spanning more than 330 kb and the presence of very long introns highlights the important contribution of LTR retrotransposons to the gene size in conifers. In contrast, the structure of the NADH-GOGAT gene is similar to the orthologous genes in angiosperms. Our phylogenetic analysis indicates that these two genes had different origins during plant evolution. The results provide new insights into the structure and molecular evolution of these essential genes.
Highlights
Plants synthesize glutamate from ammonium by the combined activity of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) through the glutamate synthase cycle
We searched for the presence of intronmediated enhancement (IME) signals that could act as enhancers of the expression along the first intron of the Fd-Glutamate synthase (GOGAT) gene, and we have found many of these short sequences in the long 50 kb first intron of the Fd-GOGAT gene
The presence of very long introns in the Fd-GOGAT gene highlights the important contribution of Long terminal repeat (LTR) retrotransposons to the gene size of conifer genes
Summary
Plants synthesize glutamate from ammonium by the combined activity of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) through the glutamate synthase cycle. Inorganic nitrogen is assimilated into the amino acids L-glutamine and L-glutamate through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Glutamate synthase (GOGAT) catalyzes the conversion of L-glutamine and 2-oxoglutarate into two molecules of L-glutamate, one of which participates in further ammonium assimilation via GS, and the other of which is used as a nitrogen donor for the production of all nitrogen-containing molecules [1]. Phylogenetic studies of nucleotide and amino acid composition have shown that genes for chloroplastic and cytosolic GS in plants come from a common ancestor that diverged before the division between angiosperms and gymnosperms [2]. The molecular analysis of GS isoenzymes in different plant species has revealed their specialization and non-overlapping roles [3, 4]
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