Minisymposium 4: Oxidative Mechanisms
Abs #
14003: 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 amylase may act on starch after cell lysis. To test this hypothesis we identified an Arabidopsis gene encoding a potentially secreted a-amylase and are using a T-DNA insertion mutant to examine starch degradation in dead tissues. Arabidopsis contains three putative a-amylase genes. One of these, AtAMY3 (At4g25000) contains an N-terminal signal sequence suggesting that it may be secreted. We obtained a mutant from the SALK Institute with a T-DNA insertion in this gene (amy3-1) and verified the presence of the insert using PCR. The mutant appears normal and is capable of transitory starch degradation. However, 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, starch-containing PAGE indicated that amylase A1 was the only amylase affected. To test the hypothesis that the secreted a-amylase degrades starch in dead cells we treated WT Arabidopsis and amy3-1 leaves with MV and observed that starch was degraded in MV-killed WT tissues but in MV-killed amy3-1 tissues, starch was not degraded over several days. In conclusion, the Arabidopsis AMY3 gene appears to encode a stress-induced, secreted a-amylase that functions in dead cells. Supported by the NSF (RUI) and the Jeffress Memorial Trust.
