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

309 Identification Of A Pyridoxine 5’-Phosphate (PNP)/Pyridoxamine 5’-Phosphate (PMP)
Oxidase (PDX3) Involved In The Vitamin B6 Salvage Pathway In Arabidopsis thaliana
Eugenia Gonzalez, Margaret Daub
North Carolina State University
PNP/PMP oxidase is a key enzyme in the formation of pyridoxal 5’-phosphate (PLP), the active coenzyme of vitamin B6,
essential in many aspects of amino acid metabolism. PLP can be synthesized de novo or by a salvage pathway. In Escherichia
coli, PNP, the direct biosynthetic intermediate of PLP, is synthesized de novo by a pathway containing two genes, PdxA
and PdxJ, and then oxidized by the PNP/PMP oxidase encoded by PdxH to form PLP. In plants, PLP is the initial product
of a different de novo pathway that involves two genes PDX1 and PDX2. In the salvage pathway in E. coli, two kinases
have been identified. PN/PM/PL kinase (PdxK) phophorylates pyridoxine (PN), pyridoxime (PM), and pyridoxal (PL) to
form PNP, PMP, and PLP, respectively. PL-specific kinase (PdxY) specifically phosphorylates PL. To date in Arabidopsis,
the only vitamin B6 salvage pathway enzyme that has been identified is a PL kinase, encoded by the salt overly sensitive
4 (SOS4) gene. In this study we identified a PNP/PMP oxidase (PDX3) involved in the vitamin B6 salvage pathway in
Arabidopsis by sequence homology. To confirm the function of this enzyme, the Arabidopsis oxidase gene (At5g49970,
renamed PDX3) was cloned and transformed into E. coli pdxH mutants that are unable to grow on minimal medium unless
amended with PL. PDX3 complemented the pdxH E. coli mutants, indicated by the ability of the transformed mutants to
grow on minimal medium without PL. Two different homozygous T-DNA insertion mutants of A. thaliana ecotype Columbia
were recovered for PDX3. Total vitamin B6 and the levels of PN, PM, PL, PMP, and PLP produced by pdx3 mutants, a
sos4 mutant and the wild type were determined by a yeast bioassay and HPLC. Surprisingly, sos4 mutants showed a 6.4-
fold increase in the amount of PLP, reflected in a 2.6-fold increase in total vitamin B6 compared to the wild type. The pdx3
mutants and the wild type plants showed similar levels of PN, PM, PL, PMP, and PLP and total vitamin B6. Regulation of
PDX1, SOS4 and PDX3 in the pdx3 and sos4 mutants and wild type was also determined by quantitative real time PCR.
SOS4 was significantly up-regulated in the pdx3 mutants and PDX3 remained unaffected in the sos4 mutants. PDX1.2 was
highly express in all mutants. In summary, we have confirmed the identity of a gene encoding an enzyme with PNP/PMP
oxidase activity involved in the vitamin B6 salvage pathway of Arabidopsis. The HPLC and gene regulation results suggest
that the vitamin B6 salvage pathway in Arabidopsis operates differently than in E. coli.
310 Trapping Enlightening crossovers(X) with Anchored Selectable markers (TEXAS Mapping)
Greg Hatlestad, Alan Lloyd
University of Texas at Austin
Map-based cloning of chemically induced mutations in Arabidopsis thaliana can be a long and tedious process. Because
crossover events occur randomly and are invisible until the DNA is analyzed this system requires vast amounts of time
and resources. One is forced to screen hundreds to thousands of plants for DNA polymorphisms to find crossover events
in useful positions near the mutation. To increase efficiency of fine mapping a mutation we have designed a system to
phenotypically identify plants with useful crossover events. The mutant line to be mapped is crossed to T-DNA lines with
known inserts in the general area of the unknown mutation. The F2 generation is screened for plants with the T-DNA's
dominant selectable marker and the homozygous recessive mutant phenotype. All progeny that pass this screen will have
an informative crossover event trapped between the T-DNA and the genetic lesion. A collection of such individuals should
have crossovers randomly distributed within the informative interval. This will allow the lesion-containing interval to
be narrowed and allow the calculation of the recombinational distance between the T-DNA and the lesion. To test this
procedure we are mapping the mutations in 2 known (ttg1-1 and ettin) and 3 unknown genes.