Poster: Oxidative stress

Abs # 85: Secreted α-amylase from Arabidopsis: Expression patterns and induction by ABA and methyl viologen

Presenter:	Sides, James M, sidesjm@jmu.edu
Authors	Sides, James M (A)   Edwards, Benjamin C (A)   Doyle, Elizabeth A (A)   Monroe, Jonathan D (A)
Affiliations:	(A): James Madison University

The Arabidopsis gene AMY3 (At4g25000) codes for an α-amylase that contains a typical signal sequence suggesting that it is either secreted or sequestered in lytic vacuoles. We hypothesize that the α-amylase acts on starch in dead cells after membrane deterioration or in cells undergoing autophagy. We obtained two T-DNA mutants in the amylase gene, amy3-1 and amy3-2, neither of which contains a normally prominent amylase activity band (A1) on native, starch-containing PAGE. Analysis of wild-type amylase A1 expression using native gels revealed that the enzyme is strongly expressed in seeds, senescing leaves, and in healthy leaves after ABA or methyl viologen treatment. In order to confirm and extend these observations we constructed transgenic plants containing an AMY3 promoter:GUS fusion. The 900 bp intergenic region 5' to the coding sequence of AMY3 was isolated using PCR and cloned in front of the GUS coding sequence in pBW18, a basta-resistant derivative of pCambia 1391Z. The resulting plasmid was transformed into Agrobacterium and then into Arabidopsis by floral spray. PCR assays confirmed the presence of the GUS gene in basta-resistant seedlings. Analysis of GUS activity in various tissues and after various stress treatments are underway.