Auxin as an intercellular signal - Intercellular Communication in Plants - page 12

1.4.3 GNOM and PIN dynamics
Significant complementary evidence for the importance of subcellular vesicle traf-
ficking in the process of PAT, and especially for its importance in plant development,
came from the analysis of the Arabidopsis mutant gnom . The gnom ( gn ) mutant (Plate
1.1G) was isolated from a screen for defects in early apical-basal patterning in Ara-
bidopsis seedlings (Mayer et al ., 1991). Embryos of gn mutants display a variety
of aberrations, including missing roots and fused or improperly placed cotyledons.
Most of these defects are reminiscent to defects observed when embryos are culti-
vated in the presence of polar auxin transport inhibitors (Hadfi et al ., 1998; Friml
et al ., 2003). The GN gene was identified as a GEF for ARF (ADP-ribosylation
factor) GTPases - essential regulators of vesicle trafficking in many organisms.
ARFs participate in the formation of transport vesicles from donor compartments
and the selection of their protein cargo. ARF proteins are present in two forms: an
active GTP-bound and an inactive GDP-bound form. Conversion of GDP-bound to
the GTP-bound form is mediated by specific GEFs (Donaldson & Jackson, 2000).
The function of ARF-GEFs such as GNOM is inhibited by BFA, which binds to an
ARF-GDP/ARF-GEF complex (Peyroche et al ., 1999). At first, it was difficult to
reconcile the biochemical function of GNOM in vesicle trafficking with the auxin-
transport-related phenotype of the gnom mutant. A detailed analysis of gn revealed
that at the subcellular level, the coordinated polar localization of PIN1 was defective
in gnom mutant embryos (Steinmann et al ., 1999). This finding, taken together with
aBFA-sensitive subcellular cycling of PIN proteins, suggested a role for GNOM
ARF-GEF in the regulation of subcellular trafficking of PAT components such as
PIN proteins (Fig. 1.4). Consistently with this, GNOM localizes to and maintains the
integrity of endosomes through which PIN proteins recycle (Geldner et al ., 2003).
To determine whether GNOM specifically controls PIN1 traffic, a single amino acid
substitution (696M to L) was introduced into the originally BFA-sensitive GNOM, to
generate a fully functional, but BFA-insensitive variant of the GNOM protein. This
allowed specifically dissecting the function of GNOM from other BFA-sensitive
trafficking steps. When plants expressing the BFA-resistant GNOM are treated with
BFA, PIN1 remains correctly localized to the cell surface (Fig. 1.4), demonstrating
direct involvement of GNOM in PIN1 recycling. In addition, BFA-resistant GNOM
renders both auxin efflux and auxin-mediated growth insensitive to BFA inhibition
(Geldner et al ., 2003). Thus, these findings directly linked a component of membrane
traffic - GNOM ARF-GEF to the PIN recycling and PAT process, highlighting the
importance of polarized trafficking in fundamental processes of plant development.

1.5 The role of auxin gradients in plant development

It remained a mystery for years how a simple molecule like auxin can influence
the variety of seemingly unrelated developmental processes such as embryonic
axis formation, organogenesis, meristem maintenance, tropisms, root and shoot
elongation, apical hook formation and others. The availability of molecular tools