Poster: Oxidative stress

Abs # 84: Secreted α-amylase from Arabidopsis: Developmental expression, cellular localization and influence on starch metabolism

Presenter:	Lane, Ashleigh M, laneam@jmu.edu
Authors	Lane, Ashleigh M (A)   Jones, Blake C (A)   Crawford, Ashley C (A)   Redick, Jaime E (A)   Doyle, Elizabeth A (A)   Monroe, Jonathan D (A)
Affiliations:	(A): James Madison University

The Arabidopsis gene AMY3 (At4g25000) encodes 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. Transitory starch metabolism in leaves appears to be unaffected by the mutation as expected given the spatial separation of starch and the amylase. 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. To extend these observations, we are currently analyzing wild type, amy3-1 and amy3-2 plants for levels of starch in developing seeds, in senescing leaves and in plants treated with methyl viologen. Phenotypically, the amy3-1 mutation has no apparent effect on seed development, germination or plant growth; however, mutant leaves exposed to high light levels during senescence appear to accumulate more anthocyanin than wild type plants, suggesting a higher susceptibility to stress in the amy3-1 mutant. To address the cellular localization of the AMY3 protein, wild type protoplasts were prepared and subjected to native, starch-containing PAGE. The protoplasts contained no detectable amylase A1 activity, indicating that the activity is localized to the apoplast in leaf cells. To further the confirm the cellular localization of the AMY3 protein, we are constructing an AMY3:GFP fusion construct using the native AMY-3 signal peptide in the vector pCAMBIA1302.