Novel aspects of glutamine synthetase (GS) regulation revealed by a detailed expression analysis of the entire GS gene family of Medicago truncatula under different physiological conditions

Abstract

Background: Glutamine Synthetase (GS, EC 6.3.1.2) is a central enzyme in nitrogen metabolism, and a key component of nitrogen use efficiency (NUE) and plant yield and thus it is extremely important to understand how it is regulated in plants. Medicago truncatula provides an excellent model system to study GS, as it contain a very simple GS gene family comprising only four expressed genes, MtGS1a and MtGS1b encoding cytosolic polypeptides, and MtGS2a and MtGS2b encoding plastid-located enzymes. To identify new regulatory mechanisms controlling GS activity, we performed a detailed expression analysis of the entire GS gene family of M. truncatula in the major organs of the plant, over a time course of nodule or seed development and during a diurnal cycle.

Results: Individual GS transcripts were quantified by qRT-PCR, and GS polypeptides and holoenzymes were evaluated by western blot and in-gel activity under native electrophoresis. These studies revealed that all four GS genes are differentially regulated in each organ of the plant, in a developmental manner, and identified new regulatory controls, which appear to be specific to certain metabolic contexts. Studies of the protein profiles showed that the GS polypeptides assemble into organ-specific protein complexes and suffer organ-specific post-translational modifications under defined physiological conditions. Our studies also reveal that GS expression and activity are modulated during a diurnal cycle. The biochemical properties of the four isoenzymes were determined and are discussed in relation to their function in the plant.

Conclusions: This work provides a comprehensive overview of GS expression and regulation in the model legume M. truncatula, contributing to a better understanding of the specific function of individual isoenzymes and to the identification of novel organ-specific post-translational mechanisms of GS regulation. We demonstrate that the GS proteins are modified and/or integrated into protein-complexes that assemble into a specific composition in particular organs of the plant. Taken together, the results presented here open new avenues to explore the regulatory mechanisms controlling GS activity in plants, a subject of major importance due to the crucial importance of the enzyme for plant growth and productivity.

Figure 1

Analysis of the expression of the entire M. truncatula GS gene family in several organs of the plant. The organs analysed include roots, de-nodulated roots from N₂-grown plants (Roots-DN), 14 day old nodules, leaves, stems, cotyledons from light (Light Cot) and from dark grown seedlings (Dark Cot), flowers, pods and green seeds. A. Quantification of MtGS1a, MtGS1b, MtGS2a and MtGS2b transcripts by qRT-PCR. GS expression was normalized to that of the housekeeping gene MtElf1-α. B. Western blot analysis of GS polypeptides using anti-Glnγ antibody [27] recognizing both the cytosolic (GS1) and plastid-located (GS2) polypeptides and a specific anti-GS2 antibody. Equal amount of protein were loaded on each lane, 30 μg for the anti-Glnγ antibody and 10 μg for the anti-GS2 antibody. C. Quantification of GS activity on soluble protein extracts. The results presented in this figure are representative of at least four biological replicates.

Figure 2

Analysis of GS holoenzyme composition by Native polyacrylamide-gel electrophoresis in several organs of the plant. The organs analysed include roots, nodules, leaves, stems, flowers, pods, green seeds, seeds 24 hours after water imbibition (24 HAI) and seedlings. Equal amounts of protein (30 μg) were loaded in each lane. In-gel GS activity is revealed by the brown staining. The native gel was Western blotted using the anti-GS2 and anti-Glnγ antibody [27]. The same membrane was first incubated with the specific anti-GS2 antibody, stripped and re-probed with the Anti-Glnγ antibody. Three major regions of GS activity are indicated in the gel. Red and dark arrows indicate GS2 or GS1 protein complexes, respectively. The images are representative of at least three independent experiments.

Figure 3

Analysis of the expression of the entire M. truncatula GS gene family during nodule development. Nodules were collected at 0, 3, 6, 10, 14, 20 and 40 days after infection. A. Quantification of MtGS1a, MtGS1b, MtGS2a and MtGS2b transcripts by qRT-PCR. GS transcript abundance was normalized to that of the housekeeping gene MtElf1-α. B. Western blot analysis of GS polypeptides using anti-Glnγ antibody recognizing both the cytosolic (GS1) and plastid-located (GS2) polypeptides [27]. 40 day old nodules were dissected into three parts, enriched in meristematic zone (Mer), nitrogen fixation zone (Fix) and senescence zone (Sen). Equal amount of protein (30 μg) were loaded on each lane. C. Quantification of GS activity on soluble protein extracts. Results are representative of at least 3 biological replicates.

Figure 4

Analysis of the expression of the entire M. truncatula GS gene family during seed development. Seeds were collected at 3, 6, 10, 14, 20, 24 and 36 days after pollination. A. Quantification of MtGS1a, MtGS1b, MtGS2a and MtGS2b transcripts by qRT-PCR. GS transcript abundance was normalized to that of the housekeeping gene MtElf1-α. B. Western blot analysis of GS polypeptides using either an anti-Glnγ antibody recognizing both the cytosolic (GS1) and plastid-located (GS2) polypeptides [27] or a specific anti-GS2 antibody. Equal amount of protein (30 μg) were loaded on each lane. C. Quantification of GS activity on soluble protein extracts. Results are representative of three biological replicates.

Figure 5

Analysis of the expression of MtGS1a, MtGS1b and MtGS2a during a diurnal cycle. Leaf samples were collected 30 minutes before or after the transition day/night and night/day and every 4 hours over a period of 24 hours. A. The graphic represents the quantification of MtGS1a, MtGS1b and MtGS2a transcripts by qRT-PCR, normalized to the housekeeping gene MtElf1-α, and the determination of total GS activity in the leaves. B. Western blot analysis using the Anti-Glnγ antibody. Equal amount of protein (30 μg) was loaded on the gel. Results are representative of two independent experiments, each using three biological replicates.

Figure 6

Kinetics of MtGS1a, MtGS1b and MtGS2a. Dependence of v_o on glutamate (A), ATP (B) and hydroxylamine (C) concentration. The “initial rate” (v₀) of the synthetase GS reaction, catalyzed by the purified His-tagged proteins is expressed as μmol of γ-glutamyl hydroxamate produced per minute.