A mutation in GDP-mannose pyrophosphorylase causes conditional hypersensitivity to ammonium, resulting in Arabidopsis root growth inhibition, altered ammonium metabolism, and hormone homeostasis

Abstract

Ascorbic acid (AA) is an antioxidant fulfilling a multitude of cellular functions. Given its pivotal role in maintaining the rate of cell growth and division in the quiescent centre of the root, it was hypothesized that the AA-deficient Arabidopsis thaliana mutants vtc1-1, vtc2-1, vtc3-1, and vtc4-1 have altered root growth. To test this hypothesis, root development was studied in the wild type and vtc mutants grown on Murashige and Skoog medium. It was discovered, however, that only the vtc1-1 mutant has strongly retarded root growth, while the other vtc mutants exhibit a wild-type root phenotype. It is demonstrated that the short-root phenotype in vtc1-1 is independent of AA deficiency and oxidative stress. Instead, vtc1-1 is conditionally hypersensitive to ammonium (NH(4)(+)). To provide new insights into the mechanism of NH(4)(+) sensitivity in vtc1-1, root development, NH(4)(+) content, glutamine synthetase (GS) activity, glutamate dehydrogenase activity, and glutamine content were assessed in wild-type and vtc1-1 mutant plants grown in the presence and absence of high NH(4)(+) and the GS inhibitor MSO. Since VTC1 encodes a GDP-mannose pyrophosphorylase, an enzyme generating GDP-mannose for AA biosynthesis and protein N-glycosylation, it was also tested whether protein N-glycosylation is affected in vtc1-1. Furthermore, since root development requires the action of a variety of hormones, it was investigated whether hormone homeostasis is linked to NH(4)(+) sensitivity in vtc1-1. Our data suggest that NH(4)(+) hypersensitivity in vtc1-1 is caused by disturbed N-glycosylation and that it is associated with auxin and ethylene homeostasis and/or nitric oxide signalling.

Fig. 1.

Simplified representation of the D-mannose/L-galactose L-ascorbic acid biosynthetic pathway in higher plants. Enzymes are (1) phosphoglucose isomerase, (2) phosphomannose isomerase (3), phosphomannose mutase (PMM), (4) GDP-mannose pyrophosphorylase (VTC1), (5) GDP-mannose-3′,5′-epimerase, (6) GDP-L-galactose phosphorylase (VTC2/VTC5), (7) L-galactose-1-phosphate phosphatase (VTC4), (8) L-galactose dehydrogenase, (9) L-galactono-1,4-lactone dehydrogenase. (Adapted from Dowdle et al., 2007.)

Fig. 2.

Physiological characterization of the wild type and vtc mutants. (A) Ascorbic acid content in root and shoot tissue of 7-d-old seedlings grown on 1× MS. Mean values ±SE of three independent replicates are shown. (B) H₂O₂ content in whole 7-d-old seedlings of the wild type and vtc mutants. Results illustrate means ±SE of three independent replicates per genotype. (C) Primary root length in 7-d-old seedlings germinated on 1× MS. Data represent means ±SE of 53–70 replicates. (D) Phenotype of 14-d-old wild type and vtc mutant seedlings grown on 1× MS. (E) Close-up of the vtc1-1 root developmental phenotype. Bar, 1 mm. (F) Primary root length of wild-type and vtc1-1 mutant plants germinated on 1× MS medium in darkness. Data represent means ±SE of 21 replicates of the wild type and 24 replicates of vtc1-1. (G) Primary root length in 7-d-old wild-type and vtc mutant plants grown on soil. Means ±SE of 5–8 replicates are shown. Asterisks indicate significant differences between individual mutants and the wild type. *P <0.05, ***P <0.001, Student's t test.

Fig. 3.

Effect of various MS media compositions on primary root development in 7-d-old wild type and vtc1-1. (A) Primary root length when plants were grown on increasing strength of MS medium. Results illustrate means ±SE of 9–23 individual seedlings per genotype and treatment. (B) Primary root length of plants grown in the absence of phosphorous (–P), ammonium nitrate (–NH₄⁺, with potassium nitrate still present) and in the absence of all nitrogen (–N, i.e. no potassium nitrate and no ammonium nitrate). Data represent means ±SE of 6–18 replicates. (C) Effect of increasing concentrations of ammonium chloride (NH₄Cl) on root growth in plants grown on 1× MS medium lacking ammonium nitrate (–NH₄⁺), but potassium nitrate still present. Mean values ±SE of 44–102 individual seedlings per genotype and treatment are shown. Asterisks indicate significant differences between mutant and wild type. *P <0.05, **P <0.01, ***P <0.001, Student's t test.

Fig. 4.

Effect of methionine sulphoximine (MSO) on root development, ammonium (NH₄⁺) content, glutamine synthetase (GS) activity, and glutamine content in whole 7-d-old seedlings of wild-type and vtc1-1 mutant plants. Plants were germinated on 1× MS in the absence and presence of ammonium nitrate (–NH₄⁺) and MSO, respectively. (A, E) Primary root length. Mean values ±SE of 147–196 independent replicates are shown. (B, F) Ammonium content g⁻¹ fresh weight. Data represent means ±SE of three independent replicates. (C, G) Mean glutamine synthetase activity of three independent replicates ±SE. (D, H) Glutamine content g⁻¹ fresh weight. Means ±SE of three independent replicates are shown. Asterisks indicate significant differences between mutant and wild type. *P <0.05, ***P <0.001, Student's t test.

Fig. 5.

Effect of the N-glycosylation inhibitor tunicamycin and ascorbic acid precursors on primary root growth in 7-d-old wild type and vtc1-1 mutants grown on 1× MS. (A) Effect of increasing concentrations of tunicamycin. Data display 7–11 replicates per genotype and treatment. (B) Effect of increasing concentrations of D-mannose. Means ±SE of 9–16 individual replicates per genotype are shown. (C) Effect of increasing concentrations of GDP-D-mannose. Results represent means ±SE of 9–14 individual replicates per genotype and treatment. (D) Effect of L-galactose. Date illustrate means ±SE of 8–11 individual seedlings per genotype. Asterisks indicate significant differences between mutant and wild type. *P <0.05, ***P <0.001, Student's t test.

Fig. 6.

Auxin (IAA, indole-3-acetic acid) content, and effect of IAA, the ethylene precursor ACC, and salicylic acid (SA) on primary root growth in 7-d-old wild-type and vtc mutant plants grown on 1× MS. (A) Total IAA content. Means ±SE of three individual replicates per genotype are shown. (B) Primary root length in the presence of IAA. Data show means ±SE of 12–35 individual seedlings. (C) Effect of increasing concentrations of ACC. Results represent means ±SE of 8–11 individual replicates per genotype and treatment. (D) Primary root growth in SA biosynthesis mutants and double mutants deficient in AA and SA. Data illustrate means ±SE of 54–108 individual seedlings per genotype. Asterisks indicate significant differences between individual mutants and the wild type. *P <0.05, ***P <0.001, Student's t test.

Fig. 7.

The role of nitric oxide (NO) in primary root development of the wild type and vtc mutants. (A) Effect of increasing concentrations of the NO donor SNP on primary root growth in 7-d-old plants grown on 1× MS medium. Data represent means ±SE of 6–12 individual seedlings per genotype and treatment. (B) NO content in the wild type and vtc1-1 in the presence of high NH₄⁺ (1× MS) and in the absence of NH₄⁺. Data represent means ±SE of three independent replicates. Asterisks indicate significant differences between the wild type and mutants. (C) Primary root growth in the presence of the specific NO scavenger cPTIO. Results show means ±SE of ten individual seedlings per genotype and treatment. (D) Relative NOS transcript levels in the presence and absence of NH₄⁺. Results display means ±SE of four biological replicates of each genotype and treatment. *P <0.05, **P <0.01, ***P <0.001, Student's t test.