| Abstract Detail
Integrative Plant Physiology Doyle, Elizabeth A. [1], Marshall, B. Grant [1], O Neil, Anne D. [1], Schrader, Meg K. [1], Schroeder, S. Nick [1], Monroe, Jonathan D. [1]. Secreted α-amylase from Arabidopsis: Expression in ABA and disease-resistance mutants, and biochemical properties. The Arabidopsis gene AMY1 (At4g25000) encodes an α-amylase that is expressed in leaves and is secreted to the apoplast. It is induced by ABA and by biotic and abiotic stress. Because leaf starch is confined to the chloroplasts, the function of a secreted amylase is enigmatic. We hypothesize that AMY1 acts on starch in dead cells after membrane deterioration, however knockout mutants have no detectable phenotype. To analyze the role of AMY1, we are 1) characterizing the expression of the AMY1 gene in signal transduction mutants and 2) characterizing both the native and heterologously expressed AMY1. We previously showed that in 5-week old abi1-1 plants, AMY1 expression was unaltered, but abi2-1 mutants showed a 34.5-fold repression of AMY1 relative to wild type. Wild type, abi1-1, and abi2-1 leaves were floated on 50 µM ABA for 24 hours and subjected to native, starch-containing PAGE. AMY1 activity was induced in wild type and in abi2-1 leaves, while abi1-1 leaves did not respond to exogenous ABA. AMY1 expression was also repressed 16.0-fold in NahG mutants. To examine the properties of the AMY1 protein, it was partially purified from ABA-treated leaves using ammonium sulfate fractionation and heat treatment at 65ºC. The enzyme’s optimum pH was 5.5 and it was stable up to 40ºC but eventually denatured at higher temperatures. A Pichia pastoris expression system was used to generate recombinant AMY1 (rAMY1) protein. The protein, located in the cell free supernatant, was partially purified using ammonium sulfate fractionation. Native, starch-containing PAGE and in vitro activity assays demonstrated rAMY1 is active. The pH optimum of rAMY1 was very similar to native AMY1, with optimal activity at a pH between 5.0 and 6.0.
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1 - James Madison University, Department of Biology, 243 Burruss Hall, MSC 7801, Harrisonburg, VA, 22807, USA
Keywords:
alpha-amylase
Abscisic Acid
abiotic and biotic stresses
abi mutants
secreted.
Presentation Type: Plant Biology Abstract
Session: P
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM
Number: P04012
Abstract ID:1333 |
