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

163 What are SCAMPS doing in plants characterization of dab5-1, a delayed abscission mutant
in Arabidopsis
Hongyu Rao 1 , Karianne Kusner 1 , Melinka Butenko 2 , Wuyi Wang 3 , Joshua Lindsey 4 , Sara Patterson 1
1
Department of Horticulture, Cellular and Molecular Biology Program, University of Wisconsin -Madison,
Madison, WI 53706, USA, 2 Department Molecular Biology, University of Oslo, Oslo Norway, 3 Ceres, Thousand
Oaks, CA , 4 Department of Surgery, Medical School, University of Wisconsin-Madison, Madison, WI 53706,
USA
The study of leaf abscission in plants was pioneered in the early 1900s by Neljubov with the observation that
ethylene gas from streetlamps caused early senescence and abscission. Subsequent studies focused on the anatomy of
the abscission zone of tomato Lycopersicum, tobacco and Sambucus, and transmission electron micrograph observations
on these abscission zones indicated that during abscission increased vesicle trafficking and Golgi activity occurred. In
following years, these studies were complemented by biochemical studies and a significant amount was ascertained
about some of the enzymes involved in cell wall degradation. Although much of the focus on abscission has focused on
cell wall associated genes, many researchers have speculated that changes in signaling in the secretory pathway may
also directly regulate the abscission process. This hypothesis is not really new, as we know that abscission is an active
process associated with increased vesicle trafficking; and consequently, it is logical to predict that mutations in genes
regulating the secretory pathway would disrupt the abscission process. Despite these predictions, many of the genes in
Arabidopsis that have been identified to have a role in vesicle trafficking have no phenotypic changes when disrupted.
Using a forward genetic screen for mutants with delayed floral organ abscission, we have identified a T-DNA insertion
upstream of a secretory membrane carrier protein (SCAMP) in dab5-1 that is responsible for delayed abscission. SCAMPs
have been studied in mammalian systems and are associated with endocytic and exocytic trafficking. We will present
characterization of the dab5-1 mutant, isolation and cloning of the T-DNA insertion, molecular complementation, and
gene expression.
164 Constructing a Gene Regulatory Network for the Arabidopsis Female Gametophyte
Jayson Punwani, David Rabiger, Gary Drews
Department of Biology, University of Utah, Salt Lake City, UT 84112
The female gametophyte (FG) plays an essential role in plant reproduction. The Arabidopsis FG is a seven-celled
structure composed of one central cell, one egg cell, two synergid cells, and three antipodal cells. How these cells acquire
their unique features and functions during development is not understood. As an entryway to dissecting the gene regulatory
networks directing cell specification and differentiation during FG development, we identified a group of genes expressed
in the Arabidopsis FG. One of the FG-expressed genes we identified, MYB98, is a member of the R2R3-MYB family of
transcription factors. The MYB98 gene is expressed in the synergid cells of the FG. myb98 FGs have defects in pollen
tube guidance and formation of the filiform apparatus [Kasahara et. al., (2005) Plant Cell 17:2981-2992]. These data
suggest that MYB98 functions as a transcription factor within the synergid cell gene regulatory network and controls the
expression of a battery of downstream genes required for pollen tube guidance and formation of the filiform apparatus.
To identify genes downstream of MYB98, we used real-time RT-PCR to screen for genes that are downregulated in myb98
FGs. We have identified more than 10 genes via this screen. Using promoter:reporter constructs, we have shown that
these genes are expressed in the synergid cells of wild-type FGs, but not myb98 FGs. To determine the cis-regulatory
elements required for expression within the synergid cells, we are dissecting the promoters of these genes. To determine
the MYB98 consensus DNA binding site, we are using the selected and amplified binding site (SAAB) assay. To ascertain
the function of these downstream genes, we are analyzing the phenotypes of mutants and determining the subcellular
localization of GFP fusion proteins.