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

Barth C, Gouzd ZA, Steele HP, Imperio RM - J. Exp. Bot. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, West Virginia University, 53 Campus Drive, Morgantown, WV 26506-6507, USA. carina.barth@mail.wvu.edu

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.

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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.
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fig5: 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.

Mentions: Increasing concentrations of tunicamycin impair root elongation in the wild type, vtc2-1, vtc3-1, and vtc4-1 mutants, but not in vtc1-1. At a concentration of 0.1 μM, these four genotypes mimicked the vtc1-1 short-root phenotype (Fig. 5A; see Supplementary Fig. S5A at JXB online), suggesting that N-glycosylation is impaired in vtc1-1. Addition of mannose did not rescue the short-root phenotype in vtc1-1. Instead, high concentrations of mannose caused an inhibition in root elongation in the wild type, while root development in vtc1-1 was unchanged (Fig. 5B). An inhibitory effect of mannose on root growth has been reported previously (Lukowitz et al., 2001). Surprisingly, GDP-mannose did not rescue the vtc1-1 root developmental phenotype (Fig. 5C), even at high concentrations. It is possible that GDP-mannose was not taken up or was unstable in the medium. As expected, galactose did not rescue the short-root phenotype in vtc1-1. Instead, it also had an inhibitory effect on root development (Fig. 5D). Similar results were found when ascorbic acid was applied to the growth medium (data not shown).


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

Barth C, Gouzd ZA, Steele HP, Imperio RM - J. Exp. Bot. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803207&req=5

fig5: 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.
Mentions: Increasing concentrations of tunicamycin impair root elongation in the wild type, vtc2-1, vtc3-1, and vtc4-1 mutants, but not in vtc1-1. At a concentration of 0.1 μM, these four genotypes mimicked the vtc1-1 short-root phenotype (Fig. 5A; see Supplementary Fig. S5A at JXB online), suggesting that N-glycosylation is impaired in vtc1-1. Addition of mannose did not rescue the short-root phenotype in vtc1-1. Instead, high concentrations of mannose caused an inhibition in root elongation in the wild type, while root development in vtc1-1 was unchanged (Fig. 5B). An inhibitory effect of mannose on root growth has been reported previously (Lukowitz et al., 2001). Surprisingly, GDP-mannose did not rescue the vtc1-1 root developmental phenotype (Fig. 5C), even at high concentrations. It is possible that GDP-mannose was not taken up or was unstable in the medium. As expected, galactose did not rescue the short-root phenotype in vtc1-1. Instead, it also had an inhibitory effect on root development (Fig. 5D). Similar results were found when ascorbic acid was applied to the growth medium (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, West Virginia University, 53 Campus Drive, Morgantown, WV 26506-6507, USA. carina.barth@mail.wvu.edu

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.

Show MeSH
Related in: MedlinePlus