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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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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.
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fig2: 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.

Mentions: The genetic defect in the vtc mutants causes AA-deficiency to a similar degree in roots and to varying degrees in shoots. All vtc mutants contained approximately 45% of the wild-type root AA content, whereas the AA content of shoots was approximately 30–50% of that of the wild type (Fig. 2A). At this early developmental stage, H2O2 content was similar in whole seedlings of the wild type and vtc mutants (Fig. 2B). Similar results were reported previously (Kotchoni et al., 2009). Nevertheless, it was predicted that root growth would be inhibited in the vtc mutants given the function of AA in the root apical meristem (Kerk and Feldman, 1994). However, root growth on full-strength MS medium (without sucrose) was only affected in vtc1-1, while the vtc2-1, vtc3-1, and vtc4-1 mutants exhibited a root developmental phenotype similar to the wild type. The vtc1-1 mutants had four times shorter primary roots than the wild type and the other vtc mutants when plants were 7 d old (Fig. 2C). Additional developmental defects became apparent in 14-d-old seedlings. Whereas formation of lateral roots was normal in the vtc2-1, vtc3-1, and vtc4-1 mutants compared to the wild type, vtc1-1 mutants initiated lateral root primordia whose elongation was strongly retarded. Furthermore, vtc1-1 mutants initiated adventitious roots at the hypocotyl–root transition zone, which were not observed in the other genotypes (Fig. 2D, E). When wild-type and vtc1-1 mutant plants were germinated on 1× MS in darkness, root development was similar in both genotypes (Fig. 2F). Finally, root development was unaffected in vtc1-1 when plants were grown on soil (Fig. 2G).


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)

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

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

fig2: 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.
Mentions: The genetic defect in the vtc mutants causes AA-deficiency to a similar degree in roots and to varying degrees in shoots. All vtc mutants contained approximately 45% of the wild-type root AA content, whereas the AA content of shoots was approximately 30–50% of that of the wild type (Fig. 2A). At this early developmental stage, H2O2 content was similar in whole seedlings of the wild type and vtc mutants (Fig. 2B). Similar results were reported previously (Kotchoni et al., 2009). Nevertheless, it was predicted that root growth would be inhibited in the vtc mutants given the function of AA in the root apical meristem (Kerk and Feldman, 1994). However, root growth on full-strength MS medium (without sucrose) was only affected in vtc1-1, while the vtc2-1, vtc3-1, and vtc4-1 mutants exhibited a root developmental phenotype similar to the wild type. The vtc1-1 mutants had four times shorter primary roots than the wild type and the other vtc mutants when plants were 7 d old (Fig. 2C). Additional developmental defects became apparent in 14-d-old seedlings. Whereas formation of lateral roots was normal in the vtc2-1, vtc3-1, and vtc4-1 mutants compared to the wild type, vtc1-1 mutants initiated lateral root primordia whose elongation was strongly retarded. Furthermore, vtc1-1 mutants initiated adventitious roots at the hypocotyl–root transition zone, which were not observed in the other genotypes (Fig. 2D, E). When wild-type and vtc1-1 mutant plants were germinated on 1× MS in darkness, root development was similar in both genotypes (Fig. 2F). Finally, root development was unaffected in vtc1-1 when plants were grown on soil (Fig. 2G).

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