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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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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.
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fig7: 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.

Mentions: The response of the wild type and vtc mutants to exogenous NO was tested and the NO content was measured. With increasing concentrations of the NO donor, SNP, primary root growth was strongly inhibited in the wild type, vtc2-1, vtc3-1, and vtc4-1 seedlings, whereas SNP had no significant effect on the already short roots in vtc1-1 (Fig. 7A). This result suggests a high endogenous NO content in vtc1-1 compared with the wild type and the other vtc mutants. The NO content in the wild type and vtc1-1 in the presence of excess NH4+ and in the absence of NH4+ was, therefore, determined. As expected, the NO content was higher in vtc1-1 when grown on 1× MS, but decreased in the absence of NH4+ (Fig. 7B). To test whether this is an NO-specific response, it was investigated whether root growth inhibition can be rescued by the addition of the specific NO scavenger cPTIO. The short-root phenotype is partially but significantly (P <0.001) recovered in vtc1-1 in the presence of cPTIO (Fig. 7C), suggesting that NO contributes, in part, to root growth inhibition in vtc1-1 in the presence of high NH4+. Finally, it was investigated whether NO is synthesized via nitric oxide synthase (NOS) by assessing NOS transcript levels in the wild type and vtc1-1 in the presence and absence of NH4+. NOS mRNA levels were approximately 2-fold higher in vtc1-1 compared to the wild type in the presence of NH4+, whereas transcript levels were the same in both genotypes in the absence of NH4+ (Fig. 7D). These data correlate nicely with the NO content (Fig. 7C) and suggest that high concentrations of NH4+ promote the formation of NO in vtc1-1 and that NO contributes, in part, to the short-root phenotype in vtc1-1.


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)

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

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

fig7: 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.
Mentions: The response of the wild type and vtc mutants to exogenous NO was tested and the NO content was measured. With increasing concentrations of the NO donor, SNP, primary root growth was strongly inhibited in the wild type, vtc2-1, vtc3-1, and vtc4-1 seedlings, whereas SNP had no significant effect on the already short roots in vtc1-1 (Fig. 7A). This result suggests a high endogenous NO content in vtc1-1 compared with the wild type and the other vtc mutants. The NO content in the wild type and vtc1-1 in the presence of excess NH4+ and in the absence of NH4+ was, therefore, determined. As expected, the NO content was higher in vtc1-1 when grown on 1× MS, but decreased in the absence of NH4+ (Fig. 7B). To test whether this is an NO-specific response, it was investigated whether root growth inhibition can be rescued by the addition of the specific NO scavenger cPTIO. The short-root phenotype is partially but significantly (P <0.001) recovered in vtc1-1 in the presence of cPTIO (Fig. 7C), suggesting that NO contributes, in part, to root growth inhibition in vtc1-1 in the presence of high NH4+. Finally, it was investigated whether NO is synthesized via nitric oxide synthase (NOS) by assessing NOS transcript levels in the wild type and vtc1-1 in the presence and absence of NH4+. NOS mRNA levels were approximately 2-fold higher in vtc1-1 compared to the wild type in the presence of NH4+, whereas transcript levels were the same in both genotypes in the absence of NH4+ (Fig. 7D). These data correlate nicely with the NO content (Fig. 7C) and suggest that high concentrations of NH4+ promote the formation of NO in vtc1-1 and that NO contributes, in part, to the short-root phenotype in vtc1-1.

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