Molecular and Physiological Analysis of Putative Amino Acid Transporters in Arabidopsis Thaliana
Dundar, Ekrem
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https://hdl.handle.net/2142/87039
Description
Title
Molecular and Physiological Analysis of Putative Amino Acid Transporters in Arabidopsis Thaliana
Author(s)
Dundar, Ekrem
Issue Date
2003
Doctoral Committee Chair(s)
Bush, Daniel R.
Department of Study
Plant Biology
Discipline
Plant Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Botany
Language
eng
Abstract
Amino acids are the currency of nitrogen exchange in plants and their physiological and molecular analysis is essential. The objective of this thesis was to identify and analyze plant amino acid transporters using the model plant Arabidopsis thaliana. A valine resistant T-DNA insertion mutant was first analyzed for this purpose. The mutant plant was isolated after screening 4900 T-DNA insertion mutants by growing them on media containing toxic (1 mM) concentration of valine (Chen, 1997). The mutant was also resistant to azaserine suggesting it was valine uptake mutant rather than a loss of feedback inhibition mutant of acetohydroxy acid synthase. Segregation analysis suggested, however, that the T-DNA insertion was not linked to the valine resistance phenotype. The analysis also revealed a poor penetrance of the valine resistance phenotype. Therefore I switched to transposon insertion mutants to analyze plant amino acid transporters. After analyzing a number transposants, I found that ten independent transposons inserted in the same gene, At2g01170. Taking advantage of this opportunity, I used these transposants to analyze the putative amino acid transporter At2g01170. First I examined the GUS expression patterns, and found a completely different GUS staining pattern for each transposant. Detailed analysis revealed that only those transposants having insertions at the 3' end of the gene yield GUS expression. A chemotropism insensitivity was also observed in mutants of At2g01170. The transposants displaying the phenotype also were those having insertions at the same (3') site of the gene, suggesting the importance of this part for the proper function of the gene. Biochemical analysis of At2g01170 was also performed using 14C-labeled amino acid transport and plate growth experiments in yeast, and revealed a bidirectional transport activity for which the gene was named BAT1 (B&barbelow;idirectional A&barbelow;mino Acid T&barbelow;ransporter 1). BAT1 import and export activity appears to be affected by pH in parallel with the age of the yeast cells. Although some bidirectional amino acid transporters from other organisms have been reported (Hosie et al., 2001; Boulland et al., 2003), BAT1 appears to be the first example from plants, and hence represents a new class of amino acid transporters in plants.
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