| Abstract Detail
Seed Biology Chen, Tingsu [1], Majee, Susmita Maitra [2], Martin, David [3], Clarke, S. [4], Downie, Bruce [5]. Identification of PIMT target proteins in Arabidopsis seeds. The orthodox seed proteome is particularly susceptible to non-enzymatic isoaspartyl (isoAsp) formation. Such damage can be repaired by PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT, EC 2.1.1.77) using S-adenosyl methionine (SAM) as a methyl donor. When both Arabidopsis PIMTs are disrupted the double mutant (pimt1-1, pimt2-1) seeds have poor vigor relative to wild type. This phenotype may arise from the dysfunction of specific proteins, crucial for seed germination, or from a general decline in the integrity of the seed proteome. We are examining these two eventualities using two techniques. The first examines the products of cloned fragments of the seed transcriptome using phage display libraries and bio-panning for those particularly susceptible to isoAsp formation or for which AtPIMT1 has high affinity. The second contrasts the double mutant seed proteome with that of the WT using on-blot methylation with the highly active human rPIMT. Four rounds of biopanning increased phage titer by 3 orders of magnitude but only in the presence of SAM. Biopanning in the presence of S-adenosylhomocysteine did not increase phage titer. An examination of the cloned fragments most frequently captured in the biopanning with SAM identified fragments of four proteins that were susceptible to isoAsp formation. Two of these have been cloned and expressed in E. coli. The recombinant proteins will be examined for the rate with which they generate isoAsp using enzyme assays and on-blot-methylation. T-DNA insertional mutants have also been isolated for two of these targets and are being tested for seed vigor. A greater understanding of this protein repair mechanism may lead to better seed pre-storage treatments to enhance longevity.
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1 - Goteborg University, Dept. of Cell and Molecular Biology, Box 462, 40530, Goteborg, Sweden
2 - University of Kentucky, Horticulture, 434 Plant Science Building, 1405 Veterans Drive, Lexington, KY 40546
3 - University of Kentucky, Horticulture, 434 Plant Science Building, 1405 Veterans Drive, Lexington, KY, 40546
4 - University of California Los Angeles, Chemistry and Biochemistry and the Molecular Biology Institute, Paul D. Boyer Hall, LA, CA, 90095
5 - University of Kentucky, Horticulture, 434 Plant Science Building, 1405 Veterans Drive, Lexington, Kentucky, 40546-0312, U.S.A.
Keywords:
protein repair.
Presentation Type: Plant Biology Abstract
Session: P
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM
Number: P29023
Abstract ID:1890 |
