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

433 The Pyrabactins: Small Molecule Agonists of the Abscisic Acid Signaling Pathway
Pauline Fung, Freeman Chow, Yang Zhao, Nicholas Provart, Sean Cutler
University of Toronto, Department of Botany
We performed a chemical genetic screen of a 10,000 member small molecule library to identify compounds that
disrupt hypocotyl cell expansion in etiolated Arabidopsis seedlings. Six compounds were identified that inhibit germination
and ~750 compounds were found to reproducibly inhibit hypocotyl growth by >20%. Two of the germination inhibitors
identified are structurally similar and examination of an analog series shows that a pyridine moiety is essential for the
activity of these compounds that we have named the Pyrabactins (for pyridine aba activation).
To delineate their mechanism of action, the effects of the Pyrabactins were examined on mutants in the GA and
ABA signaling and biosynthetic pathways. These experiments demonstrate that Pyrabactins require a functional ABA
signaling, but not biosynthetic, pathway to inhibit germination suggesting that Pyrabactins activate the ABA signaling
pathway. To examine this hypothesis further, whole genome transcript profiling was used and these experiments reveal
that ABA and Pyrabactin treatments induce strikingly similar expression profiles. Moreover, to isolate ABA-specific probe
sets from germination responsive ones, transcripts that were significantly regulated by multiple germination inhibitors
were identified and excluded in our microarray analysis. Collectively our genetic, physiological and transcriptional data
suggest that the Pyrabactins inhibit germination by activating the ABA signaling pathway and thus define a new class of
synthetic plant growth regulators.
As first steps towards target identification, we have taken two approaches. A natural variation screen has identified
several strains hypersensitive to Pyrabactin A and analysis of the Cold Spring Harbor Lab ecotype (CSHL1) has shown
that it carries a recessive mutation in a locus designated POD1 (Pyrabactin overdoser) that segregates as a Mendelian
trait and maps to the bottom arm of chromosome three; map based cloning of the POD1 locus is ongoing. Additionally
an EMS screen for Pyrabactin A resistance has identified several mutations; of current interest is a dominant mutation in
PYR1 (Pyrabactin resistant) that maps to the bottom arm of chromosome 4 and is currently being fine mapped.
Our discovery of the Pyrabactins illustrates the successful application of forward chemical genetics in identifying
new plant growth regulators. Furthermore, the Pyrabactins should be useful tools for dissection and manipulation of the
ABA signaling pathway.
434 Experimental Validation of a Predicted Feedback Loop in the Circadian Gene Network of
Arabidopsis
Peter Gould 1 , James Locke 2 , Andrew Millar 3 , Anthony Hall 1
1
University of Liverpool, 2 Warwick University, 3 University of Edinburgh
Circadian rhythms, controlled by an endogenous circadian clock, have been conserved through evolution from
prokaryotes to eukaryotes. Circadian rhythms not only provide organisms with the ability to prepare for environmental
changes caused by sunset and sunrise, but also allow seasonality to be determined, an advantage for flowering in plants
and mating and hibernation in animals.
The circadian clock generates rhythms with a period length of ~ 24 hours in a diverse range of processes from
developmental to behavioural activities. Although a large percentage of genes in higher eukaryotes show circadian
rhythmic expression (5-15% of genes), only a small number of these genes are known to be involved in the circadian
clocks central network of interlocked feedback loops.
The central network in Arabidopsis thaliana has been shown to comprise of a feedback loop in which LATE
ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), repress the expression of
TIMING OF CAB EXPRESSION 1 (TOC1) which is their transcriptional activator. Although this clock mechanism
mathematically is able to produce an oscillation it cannot fit with all the experimental data to date. For instance, the
model would predict that the cca1; lhy double mutant would be arrhythmic but this is not the case. From experimental
data the cca1; lhy double mutant infact has a short period length rhythm that persists over several days.
From mathematical modelling and experimental work we present evidence for the existence of multiple feedback
loops in the Arabidopsis central network. Within this network GIGANTEA (GI) has been predicted to act as a component
of one of the interlocking loops that is critical for the working of the circadian clock. Through experimental work we
have identified a role for GI within this model.