75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

249 Application of ABC transporters to enhanced arsenic detoxification
Melissa Pischke, Richard Meagher
Genetics Department, University of Georgia, Athens, GA 30602
The World Health Organization (WHO) recognizes arsenic as a carcinogen and a serious threat to millions of people.
While arsenic occurs naturally in the earth's crust, both natural and anthropogenic activities have contributed to arsenic
mobilization and increased concentration in the environment, such that WHO guidelines for inorganic arsenic levels are
exceeded at many locations, worldwide. One mechanism cells use to deal with exposure to toxicants such as arsenic, is
to pump them out of the cytoplasm using a special type of ABC transporter called a multidrug resistance protein (MRP).
The yeast MRP, YCF1, actively pumps glutathione:arsenite conjugates into the vacuole, resulting in arsenic resistance.
A YCF1 deletion strain lacks pump activity and is hypersensitive to arsenic. We propose that enhanced versions of the
YCF1 protein will confer increased arsenic uptake, accumulation, and resistance upon transgenic cells, and the ycf1
deletion strain will provide a convenient experimental system to test this hypothesis. Our short-term goal is identification
of evolutionarily conserved residues and domains within MRPs that regulate arsenic toxicity. Our long-term goal is
environmental arsenic remediation, leading to a worldwide decrease in arsenicosis. Progress toward the following specific
aims will be discussed. (1) Enhance the arsenic-transporting pump activity of YCF1 through PCR mutagenesis. (2)
Explore the effects of corresponding mutations on human and Arabidopsis YCF1 homologs. (3) Demonstrate a mutant
MRP-mediated increase in plant arsenic resistance, sequestration, and accumulation. Our work will identify MRPs with
improved arsenic pump activity. These proteins will be good candidates for use in the phytoremediation of arsenicals.
In addition, because YCF1 activity contributes not only to arsenic, but also to cadmium, mercury, and lead resistance in
yeast, our results will likely be applicable toward remediation of several environmental toxicants.
250 Investigation of Vitamin B6 synthesis in Arabidopsis thaliana and its role in abiotic stress
response
Elizabeth Rueschhoff, Eugenia Gonzalez, Margaret Daub
North Carolina State University
Vitamin B6 (pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives) is a required cofactor
for numerous enzymatic reactions, and has recently been implicated in cellular oxidative stress defense. The de novo
pathway for biosynthesis in higher plants differs from the well-characterized pathway in E. coli and has only recently
been characterized. In addition to the de novo pathway, all organisms contain a salvage pathway that functions to
interconvert between the different vitamers. In plants, two genes are involved in the de novo pathway, PDX1 and PDX2.
In Arabidopsis there are three PDX1 homologs; PDX1.1 on chromosome 2, PDX1.2 on chromosome 3 and PDX1.3 on
chromosome 5. PDX2 exits as a single copy located on chromosome 5. PDX1 and PDX2 form a complex and function
as a glutamine amidotransferase and also carry out the ring closure step in the pathway. Thus far, our lab and others
have identified three genes in the salvage pathway; these are genes encoding a pyridoxal kinase (SOS4), a pyridoxine
phosphatase/pyridoxamine phosphatase (PNP/PMP) oxidase (PDX3), and a putative pyridoxal reductase. The goal
of this work was to investigate the effect of mutations in the Arabidopsis de novo and salvage pathways on oxidative
stress responses. Homozygous T-DNA insertion mutants have thus far been recovered for PDX1.2, PDX1.3, PDX3, and
the putative pyridoxal reductase. In addition, a sos4 mutant was kindly provided by Dr. Jian-Kang Zhu (University of
California–Riverside). Analysis of total B6 levels by HPLC and a yeast bioassay showed that levels were unaffected in
the pdx1.2 and pdx3 mutants but were 67% lower in the pdx1.3 mutant. Surprisingly, total B6 levels in the sos4 mutant
were 264% of wild type levels. Osmotic and salt stress experiments were carried out with all the mutants. Only the sos4
mutant showed significantly increased sensitivity to salt, with an 88% decrease in root growth when grown on medium
containing 100mM NaCl as compared to wild type. In addition, both the pdx1.3 and sos4 mutants showed 77% and 82%
decrease in root length, respectively, when grown on medium containing sucrose. None of the mutants showed increased
sensitivity to mannitol as compared to wild type plants. Experiments are currently underway to determine if any of the
mutants show susceptibility to environmental stresses, such as drought, high light and chilling. We are also making hybrids
between mutants to investigate the phenotype and stress responses of lines mutant in multiple B6 biosynthetic genes.