Addition of auxin prevents the loss of EIR1/PIN2 protein and PIN2-GFP fusions (Abas
et al. 2006). Similarly, in gravity-stimulated roots, the loss of PIN2-GFP on the upper side
of the root can be detected with kinetics that parallel the gravitropic response (Abas et al.
2006). Together, these results suggest that efflux carriers are regulated at the level of syn-
thesis, breakdown, and localization.
3.6.3 Regulation of auxin transport by reversible protein phosphorylation
The activity of many highly regulated proteins is controlled by reversible phosphorylation.
Therefore, it is not surprising that changes in localization and/or activity of auxin transport
proteins due to protein phosphorylation may also regulate auxin transport (as reviewed in
DeLong et al. 2002; Muday et al. 2003 ). Inhibitor studies have implicated protein kinases
in regulating auxin transport and its sensitivity to auxin transport inhibitors (Bernasconi
1996; Delbarre et al. 1998). Genetic evidence for phosphorylation control of auxin trans-
port comes from studies of the pinoid(pid) and roots curl in NPA1 (rcn1)mutants, which
have defects in genes encoding a protein kinase and a protein phosphatase regulatory sub-
unit, respectively (as reviewed in DeLong et al. 2002). The PIDgene encodes a member of
the AGC-family of serine/threonine kinases (Christensen et al. 2000), and pidmutants ex-
hibit altered auxin transport in inflorescences and a floral development defect resembling
that of the pin1mutant (Bennett et al. 1995; Christensen et al. 2000; Benjamins et al. 2001).
Overexpression of PIDreduced elongation of roots and hypocotyls, DR5:GUSexpression
in the root tip, gravitropism, and lateral root initiation (Christensen et al. 2000; Benjamins
et al. 2001). Additionally, the main root meristem was also found to collapse after a few
days of germination, followed by the emergence of lateral roots (Benjamins et al. 2001).
Treatments with the IAA efflux inhibitors, NPA and TIBA, increased root elongation
and prevented the collapse of the primary root meristem, suggesting that auxin transport
is increased in 35S::PIDseedlings and auxin transport inhibitors help to reduce auxin
transport to normal levels in these seedlings. Finally, tissue-specific overexpression of
PIDin the shoot led to increased lateral root initiation, which was blocked by the appli-
cation of NPA at the root shoot junction, consistent with PID regulating auxin flow from
the shoot into the root (Benjamins et al. 2001). Consistent with this finding, overexpres-
sion of PID in root hair and tobacco cells enhanced auxin efflux (Lee and Cho 2006). In
inflorescences,pinoidloss-of-function and PINOIDoverexpression have been suggested
to have opposite effects on the polar targeting of the PIN1 auxin efflux facilitator protein
(Friml et al. 2004), consistent with the hypothesis that reversible protein phosphorylation
by PID may act at the level of protein targeting (Muday and Murphy 2002).
Analysis of the rcn1mutant has shown that protein phosphatase 2A (PP2A) activity
regulates root auxin transport and gravitropic curvature. The RCN1gene encodes a reg-
ulatory A subunit of PP2A, and the rcn1mutant has reduced PP2A activity in vivoandin
vitro(Garbers et al. 1996; Deruère et al. 1999; Muday et al. 2006). Roots of rcn1seed-
lings have elevated basipetal auxin transport and exhibit a significant delay in gravitro-
pism (Rashotte et al. 2001). Reduced PP2A activity causes the phenotypes observed in
rcn1roots and hypocotyls because these effects can be mimicked by treating wild-type
seedlings with low doses of protein phosphatase inhibitors (Deruère et al. 1999; Rashotte
et al. 2001; Larsen and Cancel 2003; Shin et al. 2005).