75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

243 Intracellular Production Of Reactive Oxygen Species During Salt Stress Is Mediated By
PtdIns-3-Kinase Regulated Endocytosis
Yehoram Leshem
Department of Plant and environmental Sciences, Institute of Life Sciences
Oxidative stress is a common response of plants to most of abiotic stresses. Here we show that during salt stress
ROS are produced in Arabidopsis root tips within minutes in response to the ionic but not the osmotic stress. ROS
production was significantly reduced in Atrboh (phox) mutants as well as by DPI, indicating NADPH Oxidase (Phox)
mechanism of ROS production. Confocal microscopy study showed that the ROS were detected in cytosolic speckles
which were suspected to be mitochondria but were found to be endosomes as detected by specific endosomal membrane
dyes. These findings suggested the involvement of endocytosis in induction of the NADPH oxidase. Moreover Z stack
analysis of a single cell showed that these endosomes were not found near the plasma membrane but rather integrated
within the tonoplast suggesting the involvement of vacuolar transport of the ROS signal. Both the confocal microscope
findings and the Atrboh mutants results suggested that intracellular vesicle trafficking regulated the induction and the
transduction of the ROS during salt stress. Indeed treatment of WT seedlings with vesicle trafficking inhibitors such
as LY and Brefeldin A abolished completely the induction of ROS and endocytosis during salt stress. Recent studies in
mammalians systems showed that initiation of vesicle budding is induced by phosphorylation of the D3' position of the
phosphatidylinositol ring by the PtdIns-3-Kinase. Inhibition of the PtdIns-3 kinase in Arabidopsis seedlings with the
fungal inhibitor wortmannin suppressed the endocytosis and reduced ROS production during salt stress. Moreover, similar
effects were detected in Arabidopsis mutants in the PtdIns-3 kinase. These processes were restored by supplementation
with exogenous PtdInsPs that contained phosphorylated D3' but not D5'. In optimal conditions the mutants looked like
wild type but after transfer to high salt they exhibited a salt-overly-sensitive phenotype. Similar results were obtained
in wild type plants treated with wortmannin or DPI, underscoring the positive signaling role of ROS. Taken together,
these results suggest that vesicle trafficking regulates the production localization and transmission of ROS which in turn
regulates the signal transduction during salt stress.
244 An Arabidopsis Subtilase Likely Functions In Salt Stress Responses Through A Mechanism
Involving Regulated Intramembrane Proteolysis (RIP)
Jianxiang Liu, Ping Che, Renu Srivastava, Stephen Howell
Plant Sciences Institute, Iowa State University, Ames IA 50011
Arabidopsis encodes over 50 subtilisin-like serine proteases (Rautengarten et al., 2005) and one of them, AtS1P
(At5g19660), is similar to mammalian site-1 proteases (S1Ps) which function in ER stress responses and cholesterol
homeostasis through a mechanism called regulated intramembrane proteolysis (RIP). Loss-of-function mutations in
AtS1P result in heightened sensitivity to salt stress and reduced level of expression of salt stress-induced genes, such
as ATHB-7, a homeodomain transcription factor. Three Arabidopsis b-ZIP transcription factors, AtbZIP17, -28 and -49,
have structural characteristics of S1P targets in ER stress responses in that they are predicted to be type II membrane
proteins with canonical S1P cleavage sites on the luminal side of the membrane. T-DNA insertion mutation in AtbZIP17,
blocks the expression of ATHB-7 in response to salt stress and also confers highly sensitivity to salt stress. The results are
consistent with a transcription factor signaling cascade in which AtS1P activates membrane associated b-ZIP transcription
factors, such as AtbZIP17, which in turn upregulate the expression of salt stress genes, such as ATHB-7. Experiments
showing the direct cleavage of transcription factor AtbZIP17 by AtS1P are underway. This system represents a salt stress
response and signaling pathway that has not been previously described in plants.