The glutamine synthetase gene family in Populus

Abstract

Background: Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth.

Results: The GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1) and 1 which codes for the choroplastic GS isoform (GS2). Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies.

Conclusions: Our data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types.

Figure 1

Distribution of GS genes in the chromosomes of Populus trichocarpa. Linkage Groups (LG) numbers are indicated. PtGS1.3-827781 is located in the unassambled Scaffold 122. Arrows indicate the 5'-3' orientation of genes. Red arrows connected by horizontal solid lines are the duplicated GS genes. White arrows connected by dotted lines are duplicated collinear genes located adjacent to the positions where the GS genes are present. White arrows connected by dashed-dotted lines are internal duplicated genes. The position of genes is marked by the numbers of bp in each LG.

Figure 2

The family of GS duplicate genes in Populus trichocarpa. Members of the family are represented as pairs of duplicated genes. The name of each pair is indicated on the right. Exons are in red, introns in black, and the UTR regions are in blue. The numbers of nucleotides are indicated for each exon and intron. Correspondence between segments is marked by vertical lines.

Figure 3

Relationships between poplar and other GS gene families in plants. Phylogenetic analyses of predicted full-length protein sequences were performed using the neighbor joining method. Tree was constructed as described. Pt: Populus trichocarpa. Os: Oryza sativa. Vv: Vitis vinifera. Sb: Sorghum bicolor. At: Arabidopsis thaliana. Ps: Pinus sylvestris. Psi: Picea sitchensis.

Figure 4

The regulatory regions of the poplar GS genes. The 5' upstream regions of GS genes are represented. Regulatory elements conserved in each pair of duplicated genes are marked in colours. The position of the ATG is marked on the right.

Figure 5

Spatial distribution of GS gene expression in poplar trees. Total RNA was extracted from different organs of 2-month-old hybrid poplar. A, meristematic apex. A1, A2 and A3, aerial sections from the top to the bottom of tree. L, leaf lamina. V, veins. P, petiole. S, stem. R1, primary root. R2, secondary root masses. Transcript levels of PtGS1.1, PtGS1.2, PtGS1.3 and PtGS2 were determined by real-time qPCR analysis as described. Expression levels are presented as relative values to reference genes (actin2 and ubiquitin). The histograms represent the mean values of three independent experiments with standard deviations.

Figure 6

Analysis of GS polypeptides in poplar trees. Proteins were extracted from different organs of 2-month-old hybrid poplar. L, leaf. S, Stem. R, root. Thirty micrograms of proteins per lane were separated by PAGE and then transferred to a nitrocellulose membrane, where the proteins were probed using a specific antibody developed against pine GS [19]. A, One dimensional analysis. B, Two dimensional analysis. Spot variants in two dimensional gel separation of GS polypeptides has been previously reported [31] which could be the result of post-translational modifications. The molecular size (kDa) of protein markers are indicated on the left. Major GS spots observed in the different experiments are marked by arrows.

Figure 7

Seasonal changes of GS gene expression in poplar trees. Total RNA was extracted from leaves, stem, buds and bark of 10-year-old hybrid poplar trees. Transcript levels of PtGS1.1, PtGS1.2, PtGS1.3 and PtGS2 were determined by real-time qPCR analysis as described. Expression levels are presented as relative values to reference genes (actin2 and ubiquitin). The histograms represent the mean values of at least three independent experiments with standard deviations.

Figure 8

Schematic representation of the spatial and seasonal regulation of the poplar GS family. The arrows and intensity of colors indicate the existence of a gradient of gene expression along the plant axis. Maximum values of gene expression in leaves, stems and roots were considered in the scheme. Global seasonal variation reflects major changes in gene expression.