Semester

Summer

Date of Graduation

2011

Document Type

Thesis

Degree Type

MS

College

Eberly College of Arts and Sciences

Department

Biology

Committee Chair

Carina Barth

Abstract

L-ascorbic acid is an important antioxidant in both plants and animals. In plants, it is important for detoxifying reactive oxygen species that are produced during photosynthesis and cellular metabolism. It also contributes to several facets of plant growth as an enzyme cofactor, signaling molecule, and a precursor to several other metabolites. It has been implicated in the control of flowering time and senescence as well as several other growth processes largely through work with the ascorbic acid-deficient vtc mutants of Arabidopsis thaliana. Biochemical and genetic experiments have identified several pathways contributing to ascorbic acid biosynthesis, with the D-mannose/L-galactose pathway predominantly responsible for the accumulation of ascorbic acid in Arabidopsis. Key enzymes in this pathway include GDPmannose pyrophosphorylase (encoded by VTC1), GDP-galactose phosphorylase (encoded by VTC2/5), and galactose-1-P phosphatase VTC4 genes.;Nitrogen is one of the crucial minerals for plant growth and often one of the most limiting in nature. Ammonium is the favored form of nitrogen taken up by plants, but excess levels lead to toxicity since ammonia (the conjugate base) can diffuse across membranes and depolarize membrane potentials. Recent work has indicated that the enzyme GDP-mannose pyrophosphorylase (VTC1 in Arabidopsis thaliana), which generates the essential nucleotide sugar GDP-mannose, important for protein N-glycosylation, plays an important role in response to ammonium. Arabidopsis mutants with defective VTC1 have stunted growth when grown in tissue culture in the presence of ammonium. We demonstrate here that the response of VTC1 to ammonium is pH-dependent and is not a result of ascorbic acid deficiency and is largely independent of the defects in protein N-glycosylation. We speculate that VTC1 activity is regulated in a pH-dependent manner and discuss our findings in the context of recent reports showing that GDP-mannose pyrophosphorylase forms oligomers necessary for optimal enzyme activity.;Currently, the Mendelian inheritance of genetic information is regarded as a core tenet in our understanding of how genetic information is passed from one generation to the next. However, recent experiments have shown that plants are able to produce progeny that are genetically unique from their parents. The genotypes of these progeny are not predictable given the laws of Mendelian inheritance and a decisive explanation as to how they arise is still not known. Often, these genetically unique progeny are disregarded as experimental errors or contaminants. However, we have isolated a novel Arabidopsis mutant, svt2, which is capable of producing genetically distinct progeny from self-pollinated plants at a persistent relatively high rate (~10% of progeny exhibit a genotype different from the parent). The svt2 mutant was isolated in a suppressor screen of the vtc1-1 mutant, which aimed to identify genes important for the ammonium sensitivity exhibited by vtc1-1. Further characterization of the isolated M0 plant revealed a genotype that was different from vtc1-1 and the wild type (Columbia-0 [Col-0] accession), but was more similar to the genotype of Landsberg erecta-0 (Ler-0). Multiple experiments ruled out possible seed or pollen contamination. Furthermore, svt2 offspring with Col-like characteristics can produce plants with Ler-like features, suggesting genotypic and phenotypic instability in svt2. We speculate that the additive stress of the chemical mutagen used to generate svt2 and the elevated oxidative stress in the vtc1-1 mutant, trigged activation of a genome restructuring event that is an inherent capability present in plants. This is supported by other studies which show plants under intense abiotic stresses can produce genotypically different progeny.;The ascorbic acid deficiency in the vtc mutants causes several pleiotropic phenotypes beyond oxidative stress, including early flowering and senescence. In a study to identify the flowering pathway that ascorbic acid interacts in and causes this phenotype, a double mutant with a defect in the flowering time gene, FCA (fca-1, in the Ler-0 background), and vtc1-1 had partially recovered ascorbic acid but maintained a delayed flowering time. This suggested interaction of FCA or one of its interacting partners in ascorbic acid biosynthesis. Further analyses indicated that FCA does not act directly affect transcription of genes within the D-mannose/ L-galactose pathway, but may act through post-transcriptional regulation. We speculate that the increased ascorbic acid in vtc1-1 fca-1 is most likely caused by the differing accession backgrounds of the two mutants. However, since vtc1-1 fca-9 and vtc4-1 fca-9 mutants (fca-9 is in the Col-0 background) exhibit significantly increased ascorbic acid compared to their respective single vtc mutants, FCA does indeed appear to play a role in ascorbic acid biosynthesis but does so through an unidentified mechanism in the D-mannose/L-galactose pathway or possibly in another biosynthetic pathway.

Download

Share

COinS