Page 244 - PC2019 Program & Proceedings

PLANT CANADA 2019

P61. Characterizing a novel protein targeting mechanism to the outer envelope of chloroplasts
*1
1
Overton, A. ; S. Chuong ; M. Smith 2
1 University of Waterloo
2 Wilfrid Laurier University

The chloroplast contains roughly 3000 proteins, 95% of which are encoded by the nuclear genome and
must be targeted to the chloroplast post-translationally. The majority of stroma targeted proteins possess
a cleavable N-terminal transit peptide (TP) which is recognized by the translocon at the outer envelope
membrane of chloroplasts (TOC complex). Recently, a TP-like sequence has been described at the C-
terminus of a subset of chloroplast outer envelope proteins (OEPs), including one of the proteins in the
TOC complex, Toc 159. This C-terminal TP-like sequence is hypothesized to represent a novel
mechanism of OEP chloroplast targeting. This research project focusses on one of the proteins in the set
of OEPs predicted to contain a C-terminal TP-like sequence, which has been named OEP15. Subcellular
localization using transient expression assays have been performed in both onion epidermal cells and
Arabidopsis thaliana protoplasts using an EGFP fusion construct to examine the subcellular localization
of OEP15. The results of these localization assays are expected to aid in understanding of the new
chloroplast outer membrane targeting pathway.

Alyssa Overton (akoverto@uwaterloo.ca)

P62. Exploring the role of the purple acid phosphatase AtPAP17 in Arabidopsis phosphate and
ROS metabolism
O'Gallagher, B. *
Queen's University

Purple acid phosphatases (PAPs) function in the recycling and acquisition of inorganic phosphate (Pi), a
limiting macronutrient that is vital for plant growth. However, mammalian PAPs expressed in white
blood cells participate in reactive oxygen species (ROS) metabolism and immunity/pathogen defense as
they are ‘moonlighting’ enzymes that exhibit significant peroxidase in addition to phosphatase activities.
Similarly, AtPAP17 (At3g17790), one of 29 predicted Arabidopsis PAP isozymes, is a 35-kDa PAP
hypothesized to function in Pi and ROS metabolism owing to its marked induction during Pi starvation,
leaf senescence, oxidative stress due to drought or excessive salinity, and fungal or bacterial pathogen-
related biotic stress. No other Arabidopsis PAP shares this unique expression profile. We are integrating
biochemical and genetic techniques to test AtPAP17’s role in Arabidopsis Pi and ROS metabolism. A 35-
kDa monomeric PAP exhibiting acid phosphatase and peroxidase activities was purified from the
intracellular fraction and cell wall (CW) extracts of Pi-starved Arabidopsis and identified as AtPAP17.
Transient expression of AtPAP17-GFP and imaging via epifluorescence microscopy confirmed AtPAP17
targeting to lytic vacuoles of Pi-deprived Arabidopsis. Loss of AtPAP17 expression in an atpap17 T-DNA
mutant did not exert an obvious impact on ability of Arabidopsis to acclimate to nutritional Pi deprivation
or to recycle Pi during leaf senescence. Work is in progress to assess oxidative stress and immune
responses of the atpap17 mutant.

Bryden O'Gallagher (13bog@queensu.ca)

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