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

209 Resolving The Function of TAS3-Generated ta-siRNA And ASYMMETRIC LEAVES2 (AS2) in
AUXIN RESPONSE FACTORS (ARFs) 2/3/4 Regulation
Irena Pekker, John Alvarez, Moti Sabban, Yuval Eshed
Weizmann Institute of Science, Israel
ARF3/ETT and its closest homolog, ARF4, are essential components of the KAN signaling pathway, which specifies
abaxial fate early in organ development. Recently three ARF genes, ARF2, ARF3 and ARF4 were shown to be a common
target of ta-siRNAs derived from the TAS3 gene. Simultaneous downregulation of ARFs 2/3/4 significantly enhanced
the ett-1 arf4-1 double mutant phenotype, demonstrating that ARF2 carries redundant functions with ARF3 and ARF4 in
establishing the abaxial fate of lateral organs. In-situ hybridization with LNA-modified DNA probe revealed an adaxial
expression pattern of TAS3-generated ta-siRNA in wild-type Arabidopsis seedlings. To evaluate the in vivo consequences
of disrupting ta-siRNA regulation, we constructed a ta-siRNA-resistant version of ARF3 (ARF3R). By sharp contrast
to nearly normal plants expressing ectopic ARF3, plants expressing ANT>>ARF3R gave rise to two to three first radial
leaves, with gradual shift to expanding asymmetric leaves with adaxial outgrowths, indicating that posttranscriptional
ARFs regulation is crucial for organ asymmetry patterning and morphogenesis. ASYMMETRIC LEAVES1 (AS1) and
AS2 genes are also responsible for establishing leaf asymmetry by specifying leaf adaxial identity. It was shown that
ectopic expression of AS2 in the leaves resulted in the replacement of the abaxial cell types with adaxial ones in a way
that mimics the kan1-2 kan2-1 and ett-1 arf4-1 double mutant plants. However, ectopic KAN couldnt complement the
asymmetry disruptions caused by AS2 overexpression. In contrast, ectopic ETT could partially complement the ectopic
AS2 phenotype. Moreover, as2 mutation enhances 35S:ETT phenotype causing development of lotus-like leaves and
adaxial outgrowths, suggesting that AS2 may act as a negative regulator of ETT and ARF4. To distinguish between
possible posttranscriptional and translational levels of AS2 regulation we applied inducible protein expression system
where AS2 was translationaly fused with Glucocorticoid Receptor (GR). We could show that 12 hours following single
DEX application AS2 leads to significant downregulation of ETT and ARF4 transcripts. We are currently trying to figure
out whether AS2 mediated ARFs transcripts decay is caused by induction of TAS3-generated ta-siRNA cleavage.
210 Indole-3- acetic acid and its Chemical Analogue 2,4-dichlorophenoxyacetic acid Evoke
Differential Responses in Arabidopsis Root Growth
Abidur Rahman 1 , Yutaka Oono 2 , Elison Blancaflor 3 , Tobias Baskin 1
1
University of Massachusetts, Amherst, MA, 2 Japan Atomic Enrgy Association, Takasaki, Japan, 3 Noble
Foundation, Ardmore, OK
Auxin plays a pivotal role in controlling plant development from embryogenesis to senescence. Although auxin action
is becoming well understood, a gap concerns the relationship between the action of the major native auxin, indole-3-
acetic acid (IAA) and that of the synthetic auxin, 2,4-dichlorophenoxy-acetic acid (2,4-D), which is used extensively as
a source of auxin because of its greater stability. In an effort to fill the gap, we have recently characterized an arabidopsis
mutant aar1, which shows a specific resistance to 2,4-D, but exhibits a wild-type response to natural auxins, IAA, indole-
3-butyric acid (IBA) and 1-naptheleneacetic acid (NAA), suggesting that the 2,4-D and IAA response pathway is at least
partially distinct. To further illuminate the differences between these two growth regulators we took both physiological
and cell biological approaches to investigate the downstream events in arabidopsis root growth. The mechanism of
auxin-induced growth inhibition was investigated by measuring rates of root elongation and of cell production. At a
concentration inhibiting elongation rate by 50%, IAA had no significant effect on cell production rate whereas 2,4-D
reduced it dramatically. The other auxins such as IBA and NAA acted more like IAA. The differential effect of IAA and
2,4-D on cell production was further supported by the expression analysis of M-phase reporter, Cyc1B::GUS, which
was inhibited by 2,4-D but not by IAA. A link between cytoskeletal organization and the auxin-induced inhibition of
cell production and cell elongation was established by monitoring the actin organization in the roots treated with these
growth regulators. Whereas IAA appeared to bundle the actin filaments, 2,4-D degraded the actin filaments extensively,
mimicking the effect of a bona fide actin inhibitor, latrunculin B (Lat B). Additionally, Lat B mimicked the 2,4-D effect
on root elongation and cell production rate. Taken together these results suggest that 2,4-D inhibitis cell production via
the actin cytoskeleton whereas the machinery for cell elongation is most sensitive to IAA.