Poster: Oxidative Stress
Abs #
148: Secreted a-amylase may function after cell death in Arabidopsis
The pathway for starch degradation in leaves is not well understood, in part because much of the starch degrading activity is located outside the chloroplast and probably plays no role in transitory starch metabolism. For example, in pea and tobacco the most abundant shoot a-amylase is secreted. Interestingly, the pea enzyme is induced by abiotic stress while the tobacco enzyme is induced by TMV infection. Since both biotic and abotic stress can lead to starch accumulation and to cell death, we reasoned that the secreted a-amylase may act on starch after cell lysis. The Arabidopsis genome contains three a-amylase-like genes but only one, AMY3 (At4g25000) contains an N-terminal signal sequence suggesting that it may be secreted. We obtained a T-DNA insertion mutant in this gene (amy3-1) and verified the presence of the insert using PCR. Native, starch-containing PAGE revealed that amy3-1 is missing an a-amylase previously identified in Arabidopsis as "amylase A1". To determine if amylase A1 is induced by oxidative stress, we treated wild-type Arabidopsis leaves with 10 mM methyl viologen (MV) in the light and observed a 2-fold increase in total amylase activity after one hour. Native gels indicate that the affected activity is amylase A1. Consistent with our hypothesis, starch is degraded in MV-killed wild type tissues while in MV-killed amy3-1 tissues, starch is not degraded. In conclusion, the Arabidopsis AMY3 gene appears to encode a secreted a-amylase that functions in dead cells. Supported by the NSF (RUI) and the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust.
