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

379 SPIKE1 is a guanine nucleotide exchange factor that positively regulates ROP small GTPases
and controls an evolutionarily conserved pathway of actin-dependent cell morphogenesis
Dipanwita Basu, Taisiya Zakharova, Eileen Mallery, Daniel Szymanski
Purdue University
During cell morphogenesis the actin cytoskeleton is dynamically reorganized in response to endogenous signals.
The recently identified WAVE-ACTIN RELATED PROTEIN (ARP) 2/3 pathway clearly illustrates this point. Activating
signals from the small GTPase ROP are thought to activate the heteromeric WAVE complex, which in turn, positively
regulates the actin filament nucleation activity of ARP2/3. However, several aspects of this signaling pathway are not
defined. For example, which of the 11 Arabidopsis ROPs feed into the WAVE-ARP2/3 pathway and which guanine
nucleotide exchange factor(s) (GEF) promote GTP-loading of ROP upstream of the WAVE-ARP2/3 We have used a
forward genetic analysis of epidermal morphogenesis to define completely, a pathway of genes from GEF to ARP2/3-
dependent actin filament nucleation. These genes play important roles in polarized growth and cell-cell adhesion in a
variety of tissues and organs. SPIKE1 (SPK1) is the lone plant gene product that contains a DHR2 domain, the defining
feature of the DOCK family of GEFs. We will present strong genetic and biochemical data indicating that the DHR2
domain of SPK1 is critical, and that DHR2 is necessary and sufficient for activation of ROP small GTPases in vivo.
Using double mutants analyses and the well known wave and arp2/3 phenotype of trichome swelling, we find that one
function of SPK1 is to positively regulate this evolutionarily conserved actin filament nucleation pathway; a pathway
that appears to utilize only a subset of ROPs. In Rho-signaling pathways, it is extremely difficult to link directly GEF
activity to specific Rho-GTP effector targets. For example, multiple ROPs and GEFs could lie between SPK1 and the
ROP-binding WAVE protein SRA1. We tested SPK1 function as a "signaling scaffold" for downstream effectors by
assaying the ability of endogenous SPK1 to interact with WAVE complex proteins. We will present several lines of
biochemical evidence that support the hypothesis that endogenous SPK1 is physically associated with WAVE complex
proteins. The SPK1-WAVE complexes may be poised to receive and transmit ROP signals with a high degree of spatial
and temporal specificity. To our knowledge this is the first example in which the in vivo function of a signaling pathway
from upstream GEF to actin filament nucleation machinery has been defined in a multicellular organism.
380 The Arabidopsis Aleurone Layer Contributes to Seed Dormancy and Responds to GA, ABA
and Nitric Oxide
Paul Bethke 1, 2 , Natsuyo Aoyama 3 , Igor Libourel 4 , David Still 3 , Russell Jones 2
1
USDA ARS Dept of Horticulture, University of Wisconsin, Madison, USA, 2 Dept. Plant and Microbial Biology,
University of California, Berkeley, USA, 3 Dept Plant Sciences, California State Polytechnic University, Pomona,
USA, 4 Michigan State University, East Lansing, USA
Seed dormancy is a complex phenomenon influenced by both genetic and environmental factors. ABA is central to the establishment
and maintenance of seed dormancy, and gibberellins are important for germination. The volatile signaling molecule nitric oxide
(NO) reduces seed dormancy in Arabidopsis. Seeds of Arabidopsis are dormant at maturity, and depending on the ecotype and
environment, dormancy loss occurs within a few weeks or after several months. Arabidopsis seeds contain an embryo enveloped in
an aleurone layer that is in turn surrounded by the testa. Imbibed Arabidopsis seeds remained dormant when the testa was removed,
but like intact seeds germinated when treated with KCN vapors or NO gas. Removal of both the testa and the aleurone layer resulted
in growth of the embryo. Aleurone cells underwent characteristic changes in ultrastructure in seeds that will germinate, and the most
obvious was a reduction in the number of vacuoles per cell. The rate of this vacuolation was greatest for the cells proximal to the
root tip. Vacuolation did not occur in dormant seeds and was prevented by ABA or the NO scavenger cPTIO. The effect of cPTIO
was overcome by GA, and GA on its own promoted the vacuolation of aleurone cells. These data suggest that the aleurone layer
is a significant determinant of Arabidopsis seed dormancy, and that this tissue is responsive to ABA, GA and NO in ways that are
consistent with the physiology of dormancy and germination in this species.
We also quantified mRNA abundance of key genes associated with dormancy and germination in the aleurone layer and embryo
of Arabidopsis seeds. Dormant seeds of ecotype C24 were imbibed with water or with the nitric oxide (NO) scavenger cPTIO,
conditions that maintain or strengthen dormancy respectively. Other seeds were treated with KCN vapors to break dormancy in an
NO-dependent manner. Embryos and aleurone layers were dissected from seeds and quantitative PCR was used to measure mRNA
abundance at 1, 24 and 48 h after imbibition for genes associated with GA biosynthesis (GA3ox1, GA30x2), GA responses (cysteine
protease), lipid metabolism (malate synthase, isocitrate lyase), and NO synthesis (nitric oxide synthase). Our data show that genes for
GA biosynthesis were strongly up-regulated in the embryos of seeds that will germinate relative to seeds that will remain dormant.
GA3ox1 and GA3ox2, however, were not expressed at detectable levels in the aleurone layer, even at times when a GA-responsive
cysteine protease was expressed. The nitric oxide synthase was expressed in both the embryo and the aleurone layer, and expression
in the aleurone layers was strongly stimulated by the NO scavenger cPTIO.