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

431 Functional analysis of ROP10 small GTPase-gated, low dose-specific genes in abscisic acid
signalling
Zeyu Xin 1 , Zhi-Liang Zheng 1, 2
1
Department of Biological Sciences, Lehman College, City University of New York, Bronx, NY 1046, 2 Plant
Sciences PhD Program, Graduate School and University Center, City University of New York, New York, NY
10016
Abscisic acid (ABA) is a key hormone that modulates both agronomically important growth and development
processes and responses to dynamic changes of biotic and abiotic environments, but our understanding of the ABA signal
transduction network remains fragmented. ROP10, a member of bona fide signaling small GTPases in Arabidopsis thaliana,
is a plasma membrane-associated protein specifically involved in the negative regulation of various ABA responses. To
dissect the ROP10-regulated ABA signaling, we analyzed the transcriptome of the low ABA dose response. Together
with molecular and bioinformatic analyses, we have revealed two groups of genes that are likely specific to low doses
of ABA. The first group consists of 80 genes that are independent of ROP10 and the second group is gated by ROP10,
including several regulatory genes such as receptor-like kinases and transcription factors. We now report the isolation
and characterization of T-DNA knockout mutants for most of the ROP10-gated genes, and have found some of them
altered ABA responses. The finding of ROP10-gated, low ABA dose-specific components in ABA signaling is novel.
This implicates that plants use this mechanism to distinguish low versus high levels of ABA and/or mild versus severe
magnitudes of stresses in order to fine tune the ABA and stress signaling network.
432 Ice Cap version 2.0: An Improved Method for High-Throughput Tissue Harvest and
Genotyping in Arabidopsis
Katie Clark 1 , Patrick Krysan 2
1
Department of Genetics, University of Wisconsin-Madison, Madison, WI 53706, USA, 2 Horticulture
Department and Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI 53706, USA
We previously developed a method named "Ice Cap" for growing Arabidopsis plants and harvesting tissue samples
in 96-well format. The name of this method reflects the fact that root tissue is captured in 96-well plates by freezing the
water in which the roots are growing. The seedlings from which the root tissue has been harvested remain viable in a
second 96-well plate, thereby allowing individual plants with a desired genotype to be propagated. We have made several
significant improvements to the Ice Cap procedure in order to make the process more robust. The cornerstone of Ice Cap
2.0 is a novel watering system that maintains a constant water level in the root growth plate. Without this watering system
it can be difficult to achieve optimal seedling growth prior to the time when the water in the root capture plate dries out.
The incorporation of this new watering system into the Ice Cap procedure also made it possible to grow the seedlings
in 96-well plates with no lid covering the seedlings. This modification allowed improved air circulation throughout the
plate, resulting in healthier seedlings. Details of the various technical improvements to Ice Cap will be described.
We have also developed an improved strategy for PCR-based genotyping of T-DNA insertion alleles as part of our
streamlined genotyping pipeline. We will describe the details of a single-tube, multi-plex PCR method for determining
if a plant is homozygous, heterozygous, or wild-type at a given locus. By using allele-specific primers carrying a generic
21-bp tag sequence, we are able to uniformly amplify PCR products in a single reaction from either or both of the two
alleles that may be present. The resulting products are then analyzed by performing SYBR green-based melt curve analysis.
The optimized protocol that we have developed constitutes a fast, simple, cheap, and accurate method for screening large
numbers of Arabidopsis plants that are segregating T-DNA mutant alleles.