Download Title : Functional study of a pectin methylesterases (PME

Title : Functional study of a pectin methylesterases (PME), At3g49220,
during heat stress response in Arabidopsis thaliana
Author: Luo Dan-Li
Adviser: Jinn, Tsung-Luo
ABSTRACT
M17
05
Plant cell wall is a highly complex structure, and pectin is a major component of the
cell wall. Pectin modification is, in general, catalysed by a large enzyme family of
pectin pectin methylesterase (PMEs) residing in the cell wall. In previous study, we
showed that PME activity was activated by heat shock in soybean, and it retains
plasma membrane integrity and co-ordinates with heat shock proteins to confer
thermotolerance. PME is encoded by 66 genes in the Arabidopsis genome. Hence, this
study used forward genetics approach to screen the PME mutant which is important
for heat shock response. By screening with 54 T-DNA insertion lines revealed two
mutants allele of At3g49220 lost acquired thermotolerance, both of survival rates
reduced to 50% as compared with wild-type plant. PME activity also increased under
heat treatment in Arabidopsis. To establish that At3g49220 gene functions as a PME,
it was cloned and expressed in Escherichia coli; however, the crude cellular extract
showed no significant increase in PME activity following induction by IPTG. The
Coomassie blue stained SDS-PAGE gels revealed that the over-expressed PME was
predominantly present in the pellet, insoluble inclusion bodies, in E. coli cells, which
night resulted from the hydrophobic transmembrane domains of At3g49220. PME
processes the modification in Golgi body for maturation before it is secreted to the
cell wall. At3g49220 was expressed in onion epidermal cell to investigate the
subcellular localization; the At3g49220-GFP signals were localized to the cytoplasm;
however Arabidopsis protoplast expressed At3g49220-GFP signals co-localized with
the Golgi apparatus marker. The different results were maybe due to the different
biological organism. Here, we confirmed that At3g49220 plays a role in heat stress,
but the mechanisms of At3g49220 in relation to the acquisition of thermotolerance
remain unclear, and additional studies are necessary to address this issue.