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

205 Identification of Genes Implicated in Arabidopsis Root Patterning using a GAL4/UAS
Activation Tagging System
Shunsuke Miyashima, Takamitsu Waki, Takashi Hashimoto, Keiji Nakajima
Grad. School Biol. Sci., NAIST
The Arabidopsis root possesses the highly organized cell pattern. This cell pattern is formed initially during
embryogenesis. After germination, stem cells in the root meristem commence stereotyped cell division sequences
that perpetuate the cell pattern. Until now, a few recessive mutants that are defective in the root patterning have been
isolated, and their causal genes were identified. However it is becoming more difficult to identify novel genes that act
in root patterning by simple mutant screening, due to genetic redundancy. To identify novel genes involved in the root
patterning, we have developed a new activation tagging system, in which Arabidopsis plants expressing a GAL4:VP16
transcription activator in a tissue-specific manner were transformed with a T-DNA containing GAL4-binding sequences
(UAS). We screened approximately 17,200 transgenic lines and isolated eight dominant mutants designated UAS-tagged
root patterning (urp), urp1D through urp8D. Genes tagged by the UAS were identified by determining T-DNA insertion
sites, followed by co-segregation and recapitulation experiments. Seven of the eight genes were found to encode putative
transcription factors belonging to the AP2, NAC, C2H2 Zinc finger, R2R3 MYB, DOF and RWP-RK families. Expression
studies indicated that some of these genes are expressed specifically in certain cell types in the root meristem region.
These observations indicated that the GAL4/UAS activation tagging system is a useful tool to identify novel genes that
have escaped from conventional mutant screenings. Arabidopsis
206 Analysis of vesicle transport system governing vascular continuity
Satoshi Naramoto 1 , Schinichiro Sawa 1 , Koji Koizumi 2 , Takashi Ueda 1 , Akihiko Nakano 1, 3 , Hiroo Fukuda 1
1
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2 Department of
Botany, University of Toronto, 3 Molecular Membrane Biology Laboratory, RIKEN
Within the leaf of an angiosperm, the vascular system is constructed in a complex network pattern. The formation of
this vein pattern has been studied as a paradigm of tissue pattern formation in plants. In order to elucidate the molecular
mechanism controlling venation pattern, van1 to van7 mutants of Arabidopsis, showing a discontinuous venation, were
isolated. In this session, we report a molecular mechanism governing vascular continuity through the analysis of the van3
and van4 mutants that have more preferential defects in vein continuity than the other van mutants.
At first, we characterized VAN3 gene that encoded an ARF-GAP protein. ARF-GAP is known to be a regulator of
budding of vesicles in intracellular compartments. Therefore, we analyzed the subcellular localization of VAN3 with
transgenic plants and suspension cells expressing both the VENUS tagged VAN3 and a GFP tagged individual organelle
markers. These analyses showed that VAN3 was localized not only on a subpopulation of TGN and unknown organelles.
Interestingly, VAN3 was not distributed uniformly on TGN but formed a distinct domain. Taken together with the finding
that VAN3 was primarily localized in Triton X-100-insoluble fractions of microsome membranes, VAN3 may reside on
a raft-like domain in TGNs.
Next, toward characterizing the function of plant ACAPs, subcellular localization analysis of VAN3 like (VAL)
proteins was performed. This analysis showed that VAL1 and VAL2 were localized on endosomes, whereas VAL3 was
localized in the cytoplasm. Expression pattern analysis of VAN3 and VALs revealed that these genes were expressed in
distinctive developmental processes. These findings suggested that plant ACAPs were functionally differentiated.
Finally, the VAN4 gene was characterized. VAN4 encodes a novel protein having no clear homology to any gene
of known functions. However, subcellular localization analysis revealed that VAN4 resided on endosomes, suggesting
its involvement of endocytosis. The polarization of PIN1 was widely known to play a critical role in determining the
venation pattern. However, based on the venation pattern of van3pin1 and van4pin1 double mutants, we found that
PIN1 functions independently from VAN3 and VAN4. Based on these results, we discussed a venation pattern formation
mechanism from the view of vesicle transport.