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

201 Phenome-Ready Unimutant Collection of the GRAS Gene Family
Mi-Hyun Lee, Miran Kim, Jung-Ok Heo, Dong Kwan Kim, Sung Oh Sohn, BoHye Kim, Nan-Ie Yu, Chae Eun Lim, Jun
Lim
Department of Bioscience and Biotechnology, KonKuk University
GRAS proteins are named after GAI, RGA, and SCR, the first three founding members. Members of the GRAS gene
family are predicted to be transcription factors and are involved in various aspects of plant growth and development. In
particular, SCRECROW (SCR) and SHOOT-ROOT (SHR) in Arabidopsis regulate asymmetric cell divisions responsible
for ground tis sue formation in the shoot and root. In the Arabidopsis genome, 33 GRAS members have been predicted.
To study the role of GRAS members in Arabidopsis, we have begun to isolate T-DNA insertion mutant lines for 33 GARS
members. As a result, a total of 76 T-DNA insertion lines for 31 GRAS members were isolated by PCR-based genotyping
method. Most of the loss-of-function mutants failed to show an obvious visible phenotype, although mutations in the
published members exhibited corresponding phenotypes as previously reported. These observations suggest that the
GRAS members encode similar proteins performing overlapping functions during plant growth and development. Since
no obvious phenotype was visible in most of the mutants, we have generated over-expression lines for the members as
well as multiple (double, triple or quadruple) mutants. Construction of over-expression lines was facilitated by Gatewaycompatible
binary vector system.
In addition, we performed real-time quantitative RT-PCR (qRT-PCR) and yeast two-hybrid (Y2H) screening to
investigate the interactions among 33 GRAS members. Current progress in the completion of phenome-ready unimutant
collection of the GRAS family will be presented.
202 Double Mutant Analyses Reveal Functional Interactions Between MDR (PGP) ABC-
Transporter Genes and also Endogenous Regulators of Auxin Transport
Daniel Lewis, Nathan Miller, Edgar Spalding
University of Wisconsin-Madison
In roots, auxin moves toward the apex (acropetally) in central cells and towards the base (basipetally) in outer cells.
Mutations in MDR1 reduce acropetal auxin transport by 80% without affecting basipetal transport. Conversely, loss of
MDR4 reduced basipetal auxin transport by 50% without an effect on acropetal transport. Thus, these two mutations allow
the influences of acropetal and basipetal auxin flows to be separated. Previosly described differential growth phenotypes
were re-analyzed with a new version of morphometric analysis software capable of judging statistical signifigance. In
order to test the interdependence of acropetal and basipetal auxin transport on root development, the mdr1mdr4 double
mutant was subjected to gravitropic and vertical growth assays. The results show that these two transport streams act
independently to modulate separate differential growth responses. Double mutant analysis was also employed to address
the functional relationship between MDR4 and flavanoids capable of inhibiting auxin transport. By combining tt4, a
mutation that blocks flavaonid synthesis, increases basipetal auxin transport, and exhibits a slower gravitropic response
with mdr4, we were able to use analysis of the gravitropic response as a means of testing weather flavanoids act through
inhibiting MDR function in vivo as well as in heterologous systems. Preliminary results indicate the double mutant is
hypertropic compared to tt4 and becomes signifigantly more curved than wild-type after 3 hours of gravistimulation.
While these data alone do not demonstrate an epistatic relationship due to the kinetic differences between mdr4 and
mdr4tt4, they support the postulation that flavaniods at least partially affect basipetal auxin transport through MDR4.