The Plant Journal (2011) 68, 110

doi: 10.1111/j.1365-313X.2011.04668.x

FEATURED ARTICLE

Type-A response regulators are required for proper root apical

meristem function through post-transcriptional regulation
of PIN auxin efflux carriers
Wenjing Zhang1, Jennifer P. C. To1, Chia-Yi Cheng1, G. Eric Schaller2 and Joseph J. Kieber1,*
Biology Department, University of North Carolina, Chapel Hill, NC 27599, USA, and
2
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA

Received 5 April 2011; revised 31 May 2011; accepted 2 June 2011; published online 21 July 2011.
*
For correspondence (fax +1 919 962 1625; e-mail jkieber@unc.edu).

SUMMARY
The phytohormones cytokinin and auxin regulate a diverse array of plant processes, often acting together
to modulate growth and development. Although much has been learned with regard to how each of these
hormones act individually, we are just beginning to understand how these signals interact to achieve an
integrated response. Previous studies indicated that exogenous cytokinin has an effect on the transcription of
several PIN efflux carriers. Here we show that disruption of type-A Arabidopsis response regulators (ARRs),
which are negative regulators of cytokinin signalling, alters the levels of PIN proteins and results in increased
sensitivity to N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport. Disruption of eight of the 10
type-A ARR genes affects root development by altering the size of the apical meristem. Furthermore, we show
that the effect of cytokinin on PIN abundance occurs primarily at the post-transcriptional level. Alterations of
PIN levels in the type-A ARR mutants result in changes in the distribution of auxin in root tips as measured by a
DR5::GFP reporter, and an altered pattern of cell division and differentiation in the stem cell niche in the root
apical meristem. Together, these data indicate that cytokinin, acting through the type-A ARRs, alters the level
of several PIN efflux carriers, and thus regulates the distribution of auxin within the root tip.
Keywords: cytokinin, auxin, PIN, two-component signaling, root development.

INTRODUCTION
Cytokinin and auxin affect diverse aspects of plant growth
and development, including cell division, and shoot and root
initiation and growth. Cytokinins were first identified by their
ability to stimulate cell division in cultured plant cells in
concert with auxin (Miller et al., 1955, 1956), and subsequently these two phytohormones have been shown to act
together in many processes (Su et al., 2011). Recently, a
model has emerged for the interaction of cytokinin and
auxin in the regulation of root meristem function (Moubayidin et al., 2009).
The cytokinin response pathway is similar to bacterial
two-component phosphorelays (To and Kieber, 2008; Argueso et al., 2010; Perilli et al., 2010). In Arabidopsis, cytokinin
binds to Arabidopsis histidine kinase (AHK) receptors,
activating their ability to transfer a phosphoryl group
to the Arabidopsis homologues of histidine phosphotransfer
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(Hpt) proteins (AHPs). The AHPs transfer the phosphoryl

group to Arabidopsis response regulators (ARRs), which
include type-A and type-B ARRs. Analyses of single and
multiple loss-of-function mutations in the AHK, AHP and
type-B ARR genes indicate that they are positive, redundant
elements in the cytokinin primary signal transduction pathway (Higuchi et al., 2004; Nishimura et al., 2004; Mason
et al., 2005; Hutchison et al., 2006; Riefler et al., 2006;
Argyros et al., 2008), and the type-A ARRs act primarily as
negative regulators (To et al., 2004). The type-A ARRs are
transcriptionally induced by cytokinin via direct activation by
the type-B ARRs (DAgostino et al., 2000; Hwang and Sheen,
2001; Sakai et al., 2001; Mason et al., 2004).
Auxin is transported through plant tissues by the regulated expression and localization of efflux and influx carriers
(Blakeslee et al., 2005), the best understood of which are the
1

2 Wenjing Zhang et al.

PIN efflux carriers (Zazmalova et al., 2007). These are
encoded by eight partially redundant genes in Arabidopsis.
The PIN proteins localize asymmetrically on the plasma
membrane to direct polar auxin transport within a tissue.
Recent studies demonstrated that exogenous cytokinin
alters the transcription of several PIN genes in the
Arabidopsis root (Dello Ioio et al., 2008; Pernisova et al.,
2009; Ruzicka et al., 2009). Auxin and cytokinin interact to
regulate the size of the root apical meristem, with cytokinin
acting to increase the rate of cell differentiation by negatively regulating PIN1, PIN3 and PIN7 expression through
induction of SHY2/IAA3, which encodes a repressor of auxin
signalling (Dello Ioio et al., 2008). Here, we examine the role
of the type-A ARRs in this process. We show that disruption
of multiple type-A ARRs leads to a decrease in the size of the
root apical meristem, consistent with previous studies with
exogenous cytokinin. We demonstrate that expression of a
subset of PINs is modulated by cytokinin primarily at the
post-transcriptional level, and that this leads to a change in
the distribution of auxin in the root tip and perturbation in
the pattern of cell division.
RESULTS
An octuple type-A ARR mutant is affected in cytokinin
response and root development
The type-A Arabidopsis response regulator (ARR) gene
family includes 10 genes grouped in five gene pairs based
on phylogenetic analysis (Figure S1a). Previously, we have
shown that three gene pairs (ARR3/ARR4, ARR5/ARR6 and
ARR8/ARR9) are expressed in the root, and that they act
partially redundantly to negatively regulate the response of
roots to exogenous cytokinin (To et al., 2004). We further
examined the expression pattern of two additional type-A
ARR genes, ARR7 and ARR15, using promoter::GFP reporters (Muller and Sheen, 2008). No insertional mutants were
available for ARR16 and ARR17 when the studies described
in this paper began, and so the remaining two type-A ARRs
were not analysed. Both ARR7 and ARR15 showed strong
expression in the root tip (Figure S1c), implying that this
gene pair may also be involved in regulation of root growth
by cytokinin. We introduced T-DNA insertional alleles for
these two genes (Figure S1b) into various multiple type-A
ARR mutant lines to generate higher-order mutants.
Expression analysis by quantitative RT-PCR indicated that
arr7 is essentially a null allele, while arr15 is a hypomorphic
allele (Figure S1d). Previous studies indicated that the arr7
arr15 double mutant was gametophyte-lethal (Leibfried et
al., 2005). Consistent with this, very few lines homozygous
for both the arr7 and arr15 mutations were obtained in the
background of other type-A ARR loss-of-function mutants.
For example, almost no quadruple homozygous mutants
were obtained from self-pollinated arr5 arr6 arr7 arr15/arr5
arr6 arr7 ARR15 plants. However, several rare lines were

obtained from these selfed plants that were homozygous for

all four type-A ARR mutations. These arr5,6,7,15 quadruple
mutants obtained were fully fertile, indicating that disruption of arr7 and arr15 results in neither gametophyte- nor
embryo-lethality. Similar results were obtained with a
higher-order arr3,4,5,6,7,8,9,15 octuple mutant. The segregation studies suggested that the arr7 and arr15 gametophytic lethality resulted from the nature of these insertional
alleles. In the heterozygous state, approximately 50% of
the pollen from both arr7 and arr15-2 is defective; in the
homozygous state, both mutants produce little defective
pollen (Figure S1e) and were fully viable and fertile. These
results suggest that these mutations most likely contain
chromosomal translocations that occurred as a result of
the T-DNA insertion events into the ARR7 and ARR15 genes
on the top (ARR7) and bottom (ARR15) arm of chromosome 1.
The role of ARR7 and ARR15 in root growth and development was analysed by examining various multiple mutant
lines. Neither the arr7 nor the arr15 single mutants showed
any substantial effect on cytokinin responsiveness or growth
and development (data not shown). The arr5,6,7,15 quadruple mutant was more sensitive to cytokinin in a root growth
assay than the arr5,6 double mutant at concentrations of
cytokinin 10 nM. Moreover, inclusion of either the arr7 or
arr15 mutation into the sextuple arr3,4,5,6,8,9 mutant further
increased the sensitivity to cytokinin (Figure 1a). These
results indicate that ARR7 and ARR15 are partially redundant
with the other type-A ARR genes with respect to a negative
role in the response of roots to cytokinin. In addition to
affecting the response to exogenous cytokinin, disruption of
ARR7 and/or ARR15 in the background of other type-A ARR
mutations strongly affected root elongation in the absence
of exogenous cytokinin (Figure 1a), suggesting that these
type-A ARRs play a role in regulating primary root growth.
Previous studies indicated that exogenous cytokinin altered
root growth by decreasing the size of the root apical
meristem (Dello Ioio et al., 2007). Consistent with this, the
root meristem size in the arr3,4,5,6,7,8,9,15 mutant was
significantly smaller than that of the wild-type at 3 days
post-germination, and this difference became more pronounced from days 5 to 7 (Figure 1b). Disruption of type-A
ARRs also led to reduced root architecture, with the
higher-order mutants displaying stronger phenotypes
(Figure 1c).
Disruption of the type-A ARRs alters the level of PIN
proteins
To determine whether altered auxin transport contributes to
the root phenotype observed in the type-A ARR mutants, we
examined the sensitivity of the mutants to the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) (Figure 1d). Consistent with previous results suggesting a link
between PIN auxin efflux carriers and cytokinin, the type-A

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Cytokinin affects PIN protein abundance 3

Figure 1. Identification and initial characterization of the arr3,4,5,6,7,8,9,15 octuple type-A arr mutant.
(a) Growth of wild-type (WT) or the indicated multiple type-A arr mutant from day 49 on MS medium supplemented with the specified concentrations of BA or
DMSO (control). Error bars represent SE (n > 30). The experiment was repeated at least twice with consistent results.
(b) Number of cells in cortex of the root meristem of wild-type and arr3,4,5,6,7,8,9,15 mutants during the first 11 days after germination. Error bars represent SD
(n > 10). The experiment was repeated at least twice with consistent results.
(c) Numbers of 1st-order (light grey) and 2nd-order (dark grey) lateral roots in wild-type and multiple type-A ARR mutants after 12 days of growth on MS medium.
Error bars represent SE (n > 20). Asterisks indicate values that were significantly different from wild-type at P < 0.05 (Students t test). The experiment was repeated
twice with consistent results.
(d) Number of 1st-order lateral roots per mm root length in wild-type and multiple type-A ARR mutants grown on MS medium supplemented with DMSO, 0.1 or
1.0 lM NPA as indicated. Error bars represent SE (n = 15). Asterisks indicate values that were significantly different from wild-type (*P < 0.1, **P < 0.05; Students
t test).

ARR mutants displayed an increased sensitivity to NPA as

measured by the effect on lateral root formation, with the
highest-order arr3,4,5,6,7,8,9,15 mutant displaying the most
enhanced sensitivity.
The altered sensitivity of the arr3,4,5,6,7,8,9,15 mutant to
NPA suggests defects in polar auxin transport, consistent
with recent results indicating that exogenous cytokinin
down-regulates PIN expression (Dello Ioio et al., 2008;
Pernisova et al., 2009; Ruzicka et al., 2009). To test this,
transgenes expressing PINGFP fusion proteins from their
endogenous promoters (PIN1::PIN1-GFP, PIN3::PIN3-GFP,
PIN4::PIN4-GFP and PIN7::PIN7-GFP) were introgressed into
the arr3,4,5,6,7,8,9,15 mutant, and the levels of PIN expression were analysed. Consistent with the hypersensitivity to
NPA, expression of multiple auxin efflux carriers was altered
in the mutant (Figure 2). In arr3,4,5,6,7,8,9,15 root tips, the
expression of PIN4GFP was greatly reduced (Figure 2c),
and expression of both PIN1GFP and PIN3GFP in the stele
was slightly reduced compared to the wild-type (Figure 2a,b). The relative PIN fluorescence in these fusion lines

was quantified (Figure S2); the levels of PIN1GFP and PIN3

GFP fluorescence were reduced approximately 20% in the
arr3,4,5,6,7,8,9,15 mutant root tips. The expression of PIN7
GFP in the stele was slightly reduced, but its expression
in the root cap was increased and expanded (Figure 2d).
To confirm these GFP fusion results, we examined the
endogenous PIN1 protein level in the root using in situ
immunocytochemistry with an anti-PIN1 antibody. Consistent with the analysis of the fusion proteins, the level of
endogenous PIN1 protein was also reduced in the root tips
of the arr3,4,5,6,7,8,9,15 mutant (Figure 3).
We next examined the effect of disruption of the type-A
ARRs on regulation of PIN expression in response to
exogenous cytokinin. The levels of PIN1GFP and PIN3GFP
responded more strongly to cytokinin in arr3,4,5,6,7,8,9,15
mutant roots, decreasing more rapidly and to a greater
extent in response to exogenous cytokinin compared to
wild-type roots. Treatment with cytokinin for 14 and 24 h
modestly reduced PIN1GFP expression in wild-type roots,
and by 48 h, expression was almost eliminated (Figures 2a

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4 Wenjing Zhang et al.

in arr3,4,5,6,7,8,9,15 mutant roots (Figure 3). Pronounced
hypersensitivity to cytokinin in the arr3,4,5,6,7,8,9,15 mutant
roots was also observed for expression of PIN3GFP in the
stele, although expression of PIN3GFP in the columella
cells of the root cap appeared almost insensitive to cytokinin
(Figures 2b and S2b). PIN4GFP expression was greatly
reduced by cytokinin in wild-type roots, but was nearly
undetectable in the arr3,4,5,6,7,8,9,15 mutant roots even in
the absence of exogenous cytokinin (Figure 2c). Finally,
expression of the PIN7GFP reporter was slightly elevated in
response to cytokinin in wild-type roots, primarily in the
stele, but cytokinin had little effect on PIN7GFP levels in the
mutant roots (Figure 2d). Together, these results indicate
that disruption of the type-A ARRs alters the basal level of
some PINs, and sensitizes the root to the effects of
exogenous cytokinin on PIN expression.
PIN function is regulated primarily by a post-transcriptional
mechanism

Figure 2. Disruption of type-A ARRs alters the level of PINGFP proteins.

(ad) Five- to 6-day-old wild-type (WT) and arr3,4,5,6,7,8,9,15 mutant seedlings expressing PIN1GFP, PIN3GFP, PIN4GFP or PIN7GFP were treated
with 5 lM BA for the specified durations, or with DMSO (control) for 24 h, as
indicated, and then imaged by confocal microscopy. Scale bar = 20 lm. The
roots were stained with 5 lM propidium iodide to visualize the outlines of the
cells.

Figure 3. Disruption of the type-A ARRs reduces the native PIN1 protein
levels. Native PIN1 protein (detected using an anti-PIN1-specific antibody) in
the root tips of wild-type (WT) and arr3,4,5,6,7,8,9,15 mutants treated with
5 lM BA or DMSO for 24 h as revealed by whole-mount immunohistochemistry (see Experimental procedures).

and S2a). In contrast, in arr3,4,5,6,7,8,9,15 mutant roots, the

level of PIN1GFP was strongly reduced 14 h after cytokinin
treatment and was nearly absent by 24 h. The level of native
PIN1 protein showed comparable cytokinin hypersensitivity

Previous studies have shown that cytokinin alters PIN function through an alteration of PIN transcript levels mediated
by the SHY2 gene (Dello Ioio et al., 2008). We therefore
examined expression of the PIN and SHY2 genes in wildtype and arr3,4,5,6,7,8,9,15 mutant root tips. RNA was
isolated from root tips of approximately 0.5 mm (including
the meristem and transition zones), and analysed using the
Nanostring nCounter analysis system, which is a highly
accurate and sensitive method of mRNA analysis based on
hybridization of RNA to fluorescently bar-coded probes
(Geiss et al., 2008). Surprisingly, the endogenous transcript
levels of the five PIN genes did not show any substantial
differences in untreated arr3,4,5,6,7,8,9,15 mutant root tips
compared to the wild-type (Figure 4a), with the possible
exception of PIN2, which showed an approximately 25%
decrease in the mutant (Figure S3). We also analysed
expression of the PIN4GFP transgene using quantitative
RT-PCR to confirm the analysis of expression of the endogenous PIN4 gene. Consistent with the nCounter analysis of
endogenous PIN4 gene expression, the level of the RNA
transcript of the PIN4GFP transgene was not reduced in the
arr3,4,5,6,7,8,9,15 mutant roots (Figure 4b), despite the
strong reduction in PIN4GFP protein levels.
We next examined the transcript level of the PIN genes in
response to cytokinin. Cytokinin treatment had little or no
effect on PIN1 transcript levels in wild-type even after 24 h
(Figure 4a). This is distinct from the strong down-regulation
of PIN1GFP fusion protein levels observed in wild-type root
tips in response to cytokinin. In the arr3,4,5,6,7,8,9,15
mutant, the transcript level of PIN1 was reduced approximately twofold 8 h after treatment with cytokinin (Figure 4a).
The endogenous PIN4 transcript level was increased by
cytokinin treatment in wild-type and arr3,4,5,6,7,8,9,15
mutant roots (Figure 4a), and the transgenic PIN4GFP
transcript level did not change significantly in response to

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Cytokinin affects PIN protein abundance 5

Figure 4. Effects of cytokinin on the level of PIN
transcripts in wild-type and arr3,4,5,6,7,8,9,15
mutant root tips.
(a) Normalized counts for PIN1, PIN3, PIN4, PIN7
and SHY2 transcripts in 0.5 mm root tips from
wild-type (WT) and arr3,4,5,6,7,8,9,15 mutant
seedlings treated with DMSO or 5 lM BA for 8 or
24 h, as determined using Nanostring nCounter
gene expression analysis. Error bars represent
SE from three biological replicates.
(b) Relative expression level of the PIN4GFP
transcript in 0.5 mm root tips of wild-type and
arr3,4,5,6,7,8,9,15 mutants treated with 5 lM BA
or DMSO for 8 or 24 h, as determined by
quantitative RT-PCR. Error bars represent SEM
from three biological replicates.

cytokinin treatment (Figure 4b). In contrast, PIN4GFP protein levels were strongly reduced in wild-type roots in
response to cytokinin (Figure 2c).
Cytokinin treatment had only modest effects on the level
of PIN7 transcripts in wild-type and mutant root tips
(Figure 4a). PIN3 transcript levels were not significantly
altered by cytokinin in wild-type root tips, but were downregulated approximately twofold in the mutant. The expression of PIN2, which was not analysed using GFP fusions, was
slightly reduced by cytokinin in wild-type roots, and this
effect was enhanced in the arr3,4,5,6,7,8,9,15 mutant (Figure S3). Surprisingly, induction of the SHY2 transcript in the
arr3,4,5,6,7,8,9,15 mutant was similar to that observed in
wild-type roots (Figure 4a), i.e., unlike the response of many
other cytokinin-responsive genes, induction of SHY2 was
not enhanced in the mutant. Together, these data suggest
that down-regulation of PIN1, PIN3 and PIN4 in response to
cytokinin in wild-type and mutant roots occurs primarily at
the post-transcriptional level, and that the hypersensitive
response in the mutant is not the result of increased
transcriptional induction of SHY2.
The distribution of the auxin response is altered in
arr3,4,5,6,7,8,9,15 mutant roots
PIN4 has been shown to play an important role in generating
an auxin sink and maintaining auxin gradients in the root

tips (Friml et al., 2002). As PIN4GFP levels were decreased

in the root tips in the arr3,4,5,6,7,8,9,15 mutant, we examined the spatial distribution of the auxin response using the
auxin-response reporter DR5::GFP. We introgressed the
DR5::GFP transgene into the arr3,4,5,6,7,8,9,15 mutant by
multiple back-crosses. The root tips of the arr3,4,5,6,7,8,9,15
mutant showed an altered pattern of DR5::GFP expression,
most notably a reduction of DR5::GFP expression in cells of
the quiescent centre (QC) (Figures 5a and S2c). This is consistent with previous studies showing that cytokinin treatment reduced DR5 expression in the QC (Ruzicka et al.,
2009). Interestingly, although DR5::GFP expression in QC
cells was reduced in the arr3,4,5,6,7,8,9,15 mutant, the
expression in columella stem cells was somewhat elevated
(Figure 5b).
The pattern of cell division in the arr3,4,5,6,7,8,9,15
mutant is altered
The distribution of auxin in the root tip plays an important
role in regulating the pattern of cell division and differentiation. PIN4 plays a critical role in this auxin-mediated patterning; the distribution of auxin is altered in pin4 mutant
roots, leading to aberrant cell division patterns, especially
near the QC (Friml et al., 2002; Blilou et al., 2005). As PIN4
expression is greatly reduced in the arr3,4,5,6,7,8,9,15
mutant, we examined the pattern of cell division in the

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6 Wenjing Zhang et al.

Figure 5. The distribution of auxin is altered in arr3,4,5,6,7,8,9,15 mutant

roots.
(a,b) Wild-type (WT) and arr3,4,5,6,7,8,9,15 mutant roots expressing an auxinresponsive reporter, DR5::GFP. The yellow arrowheads indicate quiescent
centre cells; the blue arrowheads indicate columella stem cells.
(a) Root tips from five-day-old untreated seedlings.
(b) Five- to 6-day-old wild-type and arr3,4,5,6,7,8,9,15 mutant roots treated
with 5 lM BA for the specified time or DMSO for 24 h as a control. Scale bar =
20 lm.
The roots were stained with 5 lM propidium iodide to visualize the outlines of
the cells, and were visualized by confocal microscopy.

mutant root. The arr3,4,5,6,7,8,9,15 mutant roots showed

cell division in the normally mitotically inactive QC cells
(Figure 6a). Furthermore, some of the columella stem cells
in the arr3,4,5,6,7,8,9,15 mutant showed signs of premature
differentiation into columella cells, as indicated by staining
for starch granules (Figure 6b). Approximately 40% of the
arr3,4,5,6,7,8,9,15 mutant seedlings showed either QC division or starch-granule staining in columella stem cells, although these two phenotypes did not always appear in the
same root.
DISCUSSION
Cytokinin has been shown to inhibit auxin transport in the
root tip by inducing expression of an auxin signalling
repressor SHY2, thus down-regulating the transcription of

several PIN auxin efflux carriers (Dello Ioio et al., 2008;

Ruzicka et al., 2009). Here, we show that type-A response
regulators are involved in the negative regulation of PINs by
cytokinin in the root tips, and that this regulation primarily
involves post-transcriptional inputs. PIN1 protein levels
decreased in wild-type root tips in response to cytokinin, and
by 48 h were nearly absent. However, the transcript level of
PIN1 was not changed in response to exogenous cytokinin,
in contrast to previous reports (Dello Ioio et al., 2008; Ruzicka et al., 2009). This difference may reflect the fact that
previous studies used 2 mm root sections (Ruzicka et al.,
2009), whereas we analysed only the 0.5 mm tip of the root,
which is more specific for the root meristem region, and thus
the transcript levels more accurately reflect gene expression
changes in the root meristem region. The discrepancy may
also reflect differences in the growth or treatment conditions. Alternatively, use of the NanoString method to analyse transcript levels, which does not involve synthesis or
amplification of cDNA, may be more accurate than the
quantitative RT-PCR method used in the previous studies.
Consistent with the effect of cytokinin on PIN1 expression,
the level of PIN4GFP protein level in the root cap was
greatly reduced in response to exogenous cytokinin. Previous studies using anti-PIN4 antibodies found high PIN4
protein levels in the root cap region (Blilou et al., 2005;
Vieten et al., 2005), slightly different from the pattern
observed with PIN4GFP. However, the transcript levels of
endogenous PIN4 and transgenic PIN4GFP were not
reduced in response to exogenous cytokinin (in fact, the
endogenous PIN4 level was slightly elevated). This, coupled
with the strong decrease in PIN4GFP protein levels, is
consistent with post-transcriptional regulation of PIN4 by
cytokinin. Together, these results suggest that cytokinin
negatively regulates the abundance of these PIN proteins in
the root tip primarily through a post-transcriptional mechanism, possibly by increasing their rate of turnover.

Figure 6. The pattern of cell division and differentiation is altered in arr3,4,5,6,7,8,9,15 mutant root tips.
(a) Five-day-old root tips of wild-type (WT) and arr3,4,5,6,7,8,9,15 mutants visualized by DIC microscopy. Yellow asterisks indicate divided quiescent centre cells. E,
endodermis; C, cortex. Note that the quiescent centre cells in arr3,4,5,6,7,8,9,15 mutant roots appear to undergo anticlinal divisions.
(b) Lugol staining of wild-type (WT) and arr3,4,5,6,7,8,9,15 mutant roots. The columella stem cells (the tier of cells below the quiescent centre) show premature
differentiation as indicated by Lugol staining for starch granules (white asterisk). Red arrowheads indicate quiescent centre cells.
(c) Percentage of roots with quiescent centre (QC) divisions or starch-staining columella stem cells (CSC) in wild-type and arr3,4,5,6,7,8,9,15 mutants. At least 50
seedlings were analysed for each phenotype.

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Cytokinin affects PIN protein abundance 7

The finding that the arr3,4,5,6,7,8,9,15 mutant has a
smaller root meristem suggests that type-A ARRs may be
involved in regulation of PIN protein levels by cytokinin.
Indeed, we found that the levels of PIN1, PIN3 and PIN4
protein were significantly reduced in the arr3,4,5,6,7,8,9,15
mutant. However, the transcript level of all three PIN genes
was not appreciably altered in arr3,4,5,6,7,8,9,15 roots,
consistent with a model in which the negative regulation
of PINs by cytokinin in the root meristem involves a posttranscriptional mechanism involving the type-A ARRs. One
model consistent with the data is that the type-A ARRs
negatively regulate turnover of the PIN proteins, and the low
level of type-A ARR gene function remaining in the
arr3,4,5,6,7,8,9,15 mutant is sensitized to exogenous cytokinin. Alternatively, the type-A ARRs may act indirectly by
mediating negative feedback on the down-regulation of PIN
proteins by cytokinin. Whichever is the case, the kinetics of
SHY2 induction by benzyladenine (BA) treatment are comparable in arr3,4,5,6,7,8,9,15 and wild-type roots, suggesting
that the amplified response of the PIN proteins to cytokinin
in this mutant does not involve the ARR1SHY2 pathway.
The PIN auxin efflux carriers have been shown to play
important roles in pattern formation by focusing the auxin
maximum in the QC and restricting the expression domain
of major determinants of the root stem cell niche (Aida et al.,
2004; Blilou et al., 2005). In the arr3,4,5,6,7,8,9,15 mutant, the
spatial pattern of DR5::GFP expression is altered, with a less
pronounced maximum in the QC cells, similar to the pin2
pin3 pin7 and pin3 pin4 pin7 triple mutants (Blilou et al.,
2005). This suggests that the type-A ARRs play a positive role
in maintaining the auxin maximum in the root tip and stem
cell activity through their regulation of PIN protein levels in
the root tips. The QC cells are mitotically inactive and play
essential roles in maintaining the stem cell fate of surrounding cells. We found that the QC cells in arr3,4,5,6,7,8,9,15
mutant roots undergo mitotic division at a higher frequency
and the columella stem cells show signs of differentiation,
suggesting that the type-A ARRs are necessary for proper QC
function. It is likely that the altered QC function in
arr3,4,5,6,7,8,9,15 roots reflects the reduced auxin response
maximum in the QC. However, we cannot exclude an
additional auxin-independent role for the type-A ARRs in
maintaining QC function. Ethylene has been shown to
induce the division of QC cells independently of auxin
(Ortega-Martinez et al., 2007). As cytokinin promotes ethylene production via the two-component signalling pathway
(Hansen et al., 2009), the QC division in arr3,4,5,6,7,8,9,15
mutants may result from increased ethylene production.
However, treatment with the ethylene inhibitor 1-methylcyclopropene (MCP) did not inhibit the aberrant QC division in
arr3,4,5,6,7,8,9,15 mutants (data not shown), suggesting that
it is probably not due to ethylene over-production.
ARR7 and ARR15 have been reported to be essential for
early embryo development, in particular for formation of the

embryonic root stem cell niche (Muller and Sheen, 2008).

Although we observed an effect of disruption of ARR7 and
ARR15 on root meristem function, disruption of both genes
is clearly not lethal. This may be because the arr15-2 allele
was used, and this allele may not be null. However, we have
also examined the arr15-1 allele used in previous studies
(Muller and Sheen, 2008), and, consistent with the analysis
here, double arr7 arr15-1 mutants are also not lethal (data
not shown). A second possibility is that the previous study
used inducible RNAi to disrupt ARR7 function, and the
inconsistency could reflect the difference between inducible
down-regulation and genetic disruption. Finally, the embryos in the previous study were analysed in vitro, and this
may affect the phenotype of the double mutant. Our results
indicate that ARR7 and ARR15 are not essential for embryo
development, although they probably play a redundant role
with other type-A ARRs in proper root meristem function.
Previously, we have shown that six Arabidopsis type-A
response regulators (ARR3, ARR4, ARR5, ARR6, ARR8 and
ARR9) are negative regulators of cytokinin signalling (To
et al., 2004). Here, we further analysed two additional type-A
ARR genes, ARR7 and ARR15. Analysis of various mutant
combinations using root cytokinin response assays indicated that both ARR7 and ARR15 are partially functionally
redundant with the other type-A ARR genes. Thus, at least
eight of the 10 type-A ARRs have overlapping roles as
negative regulators of cytokinin signalling. The finding that
the arr3,4,5,6,7,8,9,15 mutant has a much shorter root than
the sextuple mutants, coupled with the observation that
ARR7 and ARR15 are highly expressed in roots, suggests
that ARR7 and ARR15, together with the other type-A ARRs,
play an important role in mediating the function of endogenous cytokinin in the root. In Arabidopsis, five pairs of typeA ARR genes appear to have arisen as a result of a
hypothesized whole-genome duplication event (To et al.,
2004), estimated to have occurred approximately 2440 million years ago (Vision et al., 2000; Blanc et al., 2003).
Population genetic theory postulates that duplicate gene
pairs will revert to a single copy over a relatively short
evolutionary timescale (Lynch and Conery, 2000). Pairs that
are retained probably have at least partially diverged in
terms of function via neofunctionalization or by evolving
distinct spatial patterns of expression. The fact that each
member of the five pairs of Arabidopsis type-A ARRs is
maintained following the most recent genome duplication
events suggests that, despite the functional redundancy of
these genes with regard to the response to exogenous
cytokinin, they probably also have non-overlapping roles.
For example, ARR3 and ARR4 have been linked to the
circadian rhythm (Salome et al., 2005), and their function in
this pathway is specifically opposed by ARR8 and ARR9.
Likewise, the ARR7 and ARR15 genes have specifically been
shown to be differentially regulated by auxin in the root and
shoot apical meristem (Muller and Sheen, 2008; Zhao et al.,

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8 Wenjing Zhang et al.

2010). The effects of cytokinin on the transcription and
stabilization of the type-A ARRs also varies in terms of the
magnitude and kinetics of induction (DAgostino et al., 2000;
To et al., 2007), suggesting that the ARRs vary in terms of
negative feedback regulation on the cytokinin signalling
pathway. We postulate that negative feedback regulation of
cytokinin signalling is the ancestral role of the type-A ARRs,
as all members appear to contribute to this function in
Arabidopsis. As more is learned regarding the function of
this gene family, unique roles will probably continue to
emerge for the individual members.
EXPERIMENTAL PROCEDURES
Plant materials
The Col-0 ecotype was used in this study. DR5::GFP (Blilou et al.,
2005), PIN1::PIN1-GFP (Benkova et al., 2003), PIN3::PIN3-GFP
(Zadnkova et al., 2010), PIN4::PIN4-GFP (Vieten et al., 2005),
PIN7::PIN7-GFP (Blilou et al., 2005), ARR7::GFP and ARR15::GFP
(Muller and Sheen, 2008) have been described previously. The arr7
and arr15-2 alleles were identified by PCR screening of pooled DNA
from T-DNA collections generated by the Arabidopsis Knockout
Facility at the University of Wisconsin-Madison (Sussman et al.,
2000) and the Salk Institute (Alonso et al., 2003), respectively. The
arr7 allele in the WS ecotype was introgressed into the Col ecotype
three times before further analysis. The insertions in arr7 and arr152 were confirmed by genomic PCR using gene-specific and T-DNA
border primers. The gene-specific PCR primers used were as follows: 5-GGCGGTTTGCAGACTCACTTACCTGA-3 and 5-GACT
CTCTCAAACATTGTCTTT-3 for ARR7, and 5-CCATTTATTCTCCTCT
CATCTC-3 and 5-ATCTAATCATCCCCATCTCC-3 for ARR15. The
T-DNA border primer used for arr7 was JL202 (5-CATTTTATAATAA
CGCTGCGGACATCTAC-3), and that for arr15 was JMLB1 (5-GGCA
ATCAGCTGTTGCCCGTCTCACTGGTG-3). The arr7 and arr15-2 alleles were crossed with arr3,4,5,6 and arr3,4,5,6,8,9, and segregants
from those crosses were genotyped and selected for further crosses
to generate arr5,6,7,15, arr3,4,5,6,7,8,9, arr3,4,5,6,8,9,15 and
arr3,4,5,6,7,8,9,15. Genotyping primers for the arr3, arr4, arr5, arr6,
arr8 and arr9 alleles are as previously described (To et al., 2004).

Plant growth conditions and phenotypic analysis

Arabidopsis seeds were surface-sterilized, cold-treated at 4C for
4 days, and grown on 1MS/1% sucrose vertical plates as previously described (To et al., 2004). Cytokinin root growth assays were
performed as previously described (To et al., 2004) with the specified media supplements. Root lengths at days 4 and 9 after germination were marked on the plates. Root elongation between days 4
and 9 was measured using the IMAGEJ program (http://rsb.info.
nih.gov/ij). A dissecting microscope was used to observe and
quantify higher-order lateral roots 12 days after germination. For
NPA response assays, lateral root primordia were quantified under
a dissecting microscope at 9 days after germination and normalized
to total root length. Root meristem size was measured as previously
described (Dello Ioio et al., 2007), except that Micropore surgical
tape (3M, http://solutions.3m.com) was used to seal the plates.

Microscopy and whole-mount immunohistochemistry

For analysis of GFP reporters in roots, intact seedlings were counterstained in propidium iodide immediately after the cytokinin treatment to visualize cell walls, and analysed using confocal scanning
laser microscopy. The GFP fluorescence and propidium iodide

staining were visualized and captured using a Zeiss LSM 510 META
scanning confocal microscope with a Zeiss Plan Neofluar 20/0.5
objective (http://www.zeiss.com/). For GFP, a 488 nm line was used
for excitation, and an emission range between 505 and 530 nm was
used for detection. For propidium iodide, a 543 nm line was used for
excitation, and a 560 nm long-pass filter was used for detection. The
wild-type and arr3,4,5,6,7,8,9,15 mutant lines harbouring each GFP
reporter construct were analysed at the same time using identical
microscope settings throughout the treatments. The GFP florescence was quantified from confocal sections that included the QC
using ImageJ (http://rsb.info.nih.gov/ij).
Whole-mount immunohistochemistry in roots was performed as
described previously (Sauer et al., 2006). The primary anti-AtPIN1
antibody (Friml et al., 2002) was used at 1:330 dilution. The
secondary CY3-conjugated anti-rabbit antibody (Jackson ImmunoResearch, http://www.jacksonimmuno.com/) was used at 1:600
dilution. The immunostained roots were visualized using a Zeiss
DUO confocal microscope with an EC Plan Neofluar 40/1.30 oilimmersion objective. A 560 nm line was used for excitation, and an
emission range between 583 and 709 nm was used for detection.
For analysis of cell division in the QC, root tips of 5-day-old
seedlings were fixed in 3:1 ethanol:acetic acid for 15 min, incubated
in 70% ethanol for another 15 min, washed in water for 2 min,
cleared in chlorohydrate solution, and observed immediately using
a Nikon E800 photomicroscope with a Nikon Plan Apo 100/1.40 oilimmersion objective (http://www.nikon.com/) using differential
interference contrast (DIC) optics. To visualize starch granules in
the root tips, 5-day-old seedlings were stained with Lugols solution
(Sigma, http://www.sigmaaldrich.com/) for 5 min, mounted on
slides with chlorohydrate solution (8:2 chlorohydrate:water), and
photographed immediately using the same Nikon DIC microscope.

RNA extraction, quantitative RT-PCR and NanoString

nCounter gene expression analysis
Seedlings were grown on MS medium and then treated with either
DMSO or BA for the indicated times. For RNA extractions, cytokinin
treatment was terminated by transferring tissue to RNAlater Solution (Ambion, http://www.ambion.com/). Root samples (the last
0.5 mm of the root tip) were collected under a Leica dissection
microscope (http://www.leica.com/), and subjected to total RNA
extraction using an RNeasy Plus kit (Qiagen, http://www.qiagen.
com/).
cDNA was prepared from the total RNA using SuperScript III
reverse transcriptase (Invitrogen, http://www.invitrogen.com/) as
described by the manufacturer. Quantitative RT-PCR was performed
using SYBR Premix Ex Taq polymerase (TaKaRa, http://www.takarabio.com/) in a DNA Engine OPTICON 2 (MJ Research, http://
www.bio-rad.com). The following primers were used: 5-AGA
GGTTGACGAGCAGATGA-3 and 5-ACCAATGAAAGTAGACGCC
A-3 for TUB4, 5-GAGCAAGGGCGAGGAGCTGTTC-3 and 5TGGTGCAGATGAACTTCAGG-3 for GFP, 5-TCTCTTCTTGTAAAGT
GACGACTG-3 and 5-TCAAATTCACCTTCAAATCCTT-3 for ARR7
(5), 5-AAACCGGTGAAGCTAGCAGA-3 and 5-TCGTTTTGAACAT
GAAGAGTCC-3 for ARR7 (3), and 5-GAGATTGCTTAAGATCTCTG
GTTG-3 and 5-CAAATCCTTAAGACCAGAAGATCC-3 for ARR15.
At least two biological samples each were analysed with three
technical replicates. The relative expression for PIN4GFP (normalized to b-tubulin as a reference gene and to the wild-type grown on
DMSO as a control sample) and standard errors were determined
using REST 2009 software (Qiagen).
The Nanostring nCounter gene expression analysis was performed as described previously (Geiss et al., 2008) by the University
of North Carolina Genomics and Bioinformatics Core Facilities
using 160 ng of total RNA extracted from 0.5 mm root tips. The

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The Plant Journal 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 110

Cytokinin affects PIN protein abundance 9

Nanostring probes targeting PIN1, PIN2, PIN3, PIN4, PIN7 and SHY2,
and the control probes for TUB4 (At5g44340), UBQ10 (At4g05320)
and APT1 (At1g27450), were designed and synthesized by
NanoString Technologies (http://www.nanostring.com). The mRNA
counts for PIN1, PIN2, PIN3, PIN4, PIN7 and SHY2 were normalized
to the reference genes TUB4, UBQ10 and APT1 as described in the
nCounter Gene Expression Assay Manual (http://www.nanostring.
com/uploads/Manual_Gene_Expression_Assay.pdf/).

ACKNOWLEDGEMENTS
We thank Tony Perdue for excellent help with confocal microscopy,
Jiri Friml (University of Gent, Department of Plant Systems Biology)
for the PINGFP lines and the PIN antibody, Bruno Muller (University of Zurich, Institute of Plant Biology) for the ARR7::GFP and
ARR15::GFP lines, and Yan Shi and Mike Topal at the University of
North Carolina Genomics Core for help with the NanoStrings analysis. This project was supported by National Science Foundation
grant IOS-1022053 to J.J.K. and G.E.S.

SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Unrooted phylogenetic tree of type-A ARRs, positions of
T-DNA insertions in arr7 and arr15 mutants, expression patterns of
ARR7 and ARR15, and pollen analysis.
Figure S2. Quantification of GFP fluorescence in the root tips of
wild-type and the arr3,4,5,6,7,8,9,15 mutant.
Figure S3. The effects of cytokinin on the level of PIN2 transcripts in
wild-type and arr3,4,5,6,7,8,9,15 mutant root tips.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.

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