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

413 Arabidopsis EER4 encodes an EIN3-interacting TAFIID transcription factor that is required
for proper response to ethylene, including induction of ERF1
Linda Robles, Jessica Wampole, Matthew Christians, Jesse Cancel, Paul Larsen
University of California-Riverside
Ethylene is a critical plant hormone that promotes the seedling triple response along with several agriculturally
important phenomena including fruit ripening and tissue senescence. Through the identification of Arabidopsis mutants
with either an ethylene insensitive or constitutive ethylene reponse phenotype, several components of the ethylene-signaling
pathway have been uncovered, although large gaps in our understanding exist. Since it is unlikely that further screening
for these mutant phenotypes will be profitable, novel approaches to identify new factors involved in this pathway are
necessary. We have focused on identification of Arabidopsis mutants with enhanced ethylene responsiveness (eer), based
on the assumption that these represent defects in factors required to dampen ethylene response. One of these mutants, eer4,
has been extensively characterized, with this loss-of-function mutation resulting in extreme exaggeration of response to
saturating levels of ethylene in the triple response assay. Molecular characterization of this mutant surprisingly revealed
ethylene insensitivity with regard to induction of the ethylene-regulated genes AtEBP, basic chitinase, and ERF1, with the
latter showing virtually no expression in eer4 leaves following ethylene treatment. Molecular cloning of the eer4 mutation
showed that it represents an inappropriate stop codon in a previously uncharacterized TAFIID transcription factor that is
ubiquitously expressed throughout the plant in an ethylene independent manner. Yeast two-hybrid and in vitro binding
assays have indicated that EER4 strongly interacts with several known components of the ethylene signaling pathway
including itself, CTR1, EIN3, and ERF1 along with the catalytic subunit of PP2a, PP2a1C. It was previously reported that
loss of PP2a activity gives an identical eer phenotype, suggesting that dephosphorylation of EER4 is required for proper
ethylene response. Based on our phenotypic, biochemical, and double mutant analyses, we propose that EER4 encodes a
factor that associates with CTR1 in the absence of ethylene and then upon ethylene perception, it is dephosphorylated and
transits to the nucleus where it recruits EIN3, ERF1, and likely other ethylene-related transcription factors for induction
of genes such as ERF1 and AtEBP along with an as yet undefined group of genes required for resetting the ethylene
response pathway following a signaling event.
414 PP2C type Phosphatase Regulates Stress-activated MAP Kinases
Alois Schweighofer, Vaiva Kazanaviciute, Irute Meskiene
Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria
PP2C type phosphatases are important regulators of signaling pathways in eukaryotes. Plants, such as Arabidopsis
contain the biggest family of PP2Cs suggesting that these phosphatases are significant in plant life processes (1). Here
a new PP2C-type phosphatase from Arabidopsis was isolated and characterized. We identified its substrates as specific
MAPKs (mitogen activated protein kinase) by yeast two-hybrid interaction approach. AP2C1 display exquisite substrate
specificity in yeast and precisely down-regulates stress activated MAPKs in planta. Activation of these MAPKs by different
cues inversely correlates to AP2C1 expression in plants and isolated cells. Inactivation of stress MAPKs depends on
the catalytic activity of AP2C1 phosphatase as indicated by introduction of loss-of-function mutation in catalytic part
of the protein. Specific ability of this phosphatase to inactivate MAPKs is demonstrated in comparison with two other
Arabidopsis PP2Cs, ABI2 and HAB1. Yeast two-hybrid screen of cDNA library with AP2C1 and analysis of knock out
and transgenic plants lines over expressing this phosphatase supports its function on stress MAPKs and propose a model of
AP2C1 action in plant cells that relates to MAPK activity control, pathogen response and ethylene production. Arabidopsis
plants with altered AP2C1 contents display compromised innate immunity, modified wound responses (notably ethylene
production) and perturbed expression patterns of defense-related genes. This is the first experimentally comprehensive
report - at the molecular and whole-organism level - of a key role for a PP2C as a MAPK phosphatase in plants.
1. Schweighofer, A., Hirt, H. and Meskiene, I. (2004) Plant PP2C phosphatases: emerging functions in stress signaling. Trends Plant Sci, 9, 236-
243.