American Journal of Plant Sciences, 2011, 2, 549-553
doi:10.4236/ajps.2011.24065 Published Online October 2011 (http://www.SciRP.org/journal/ajps)
549
ARR5 and ARR6 Mediate Tissue Specific
Cross-talk between Auxin and Cytokinin in
Arabidopsis
Aparna Kakani, Zhaohua Peng
Department of Biochemistry and Molecular Biology, Mississippi State University, Starkville, USA.
Received July 23rd, 2011; revised September 1st, 2011; accepted September 10th, 2011.
ABSTRACT
Auxin and cytokinin interaction plays an essential role in a wide range of plant growth and developmental processes.
The interaction consequence of the two hormones is highly tissue specific. The molecular mechanisms underlying the
tissue specificity are largely unknown. Here we show that the cytokinin signaling pathway key components ARR5 and
ARR6 respond to auxin with a highly tissue specific and contrasted pattern in Arabidopsis seedlings and calli in the
presence of cytokinin. Our results suggest that the two highly homologous but functionally distinct genes, ARR5 and
ARR6, play a critical role in mediating tissue sp ecific interaction between auxin and cytokinin.
Keywords: ARR5, ARR6, Auxin, Cytokinin, Callus, Arabidopsis, Auxin and Cyto kinin Interaction
1. Introduction
The interplay between auxin and cytokinin is essential
for plant growth and development. Their interactions
includes synergistic, antagonistic and additive, depending
on plant species and tissues [1-5]. Apical dominance is a
classic example of auxin and cytokinin antagonistic in-
teraction. While auxin produced at the apex represses the
outgrowth of lateral buds, cytokinin applied to lateral
buds promotes the release of lateral buds from apical
dominance. Interestingly, auxin transported from apex
promotes lateral root initiation and growth and the cyto-
kinin produced in the root cap stimulates root apical
dominance [6]. These observations suggest that interac-
tion between auxin and cytokinin is highly tissue spe-
cific.
It has been shown that removal of the endogenous
auxin source via decapitation leads to up to 40 fold in-
crease of cytokinin in xylem exudates [7,8]. Studies in
Arabidopsis demonstrate that auxin mediates a very rapid
negative control of the cytokinin pool by mainly sup-
pressing its biosynthesis via the isopentenyladenosine-5’-
mono phosphate independent pathway [9]. In addition,
auxin has been found to stimulate both oxidative break-
down and glucosylation of active cytokinins in a tissue-
dependent manner [1]. On the other hand, an increase in
free IAA (active form) has been observed both in cyto-
kinin overproducing lines of Nicotiana glutinosa trans-
formed with the bacterial cytokinin biosynthesis gene ipt
(isopentenyl transferase) and in maize and pea treated
with exogenously applied cytokinin [10-12], probably via
cytokinin inhibition of enzymes that conjugate free IAA
[13]. Meanwhile, it has also been reported that cytokinin
overexpression lead to down regulation of the IAA pool
in tobacco [14]. The discrepancy among different studies
suggests that further investigations are required for a
complete picture of the auxin-cytokinin interaction.
The auxin and cytokinin control of cell division in un-
differentiated cells presents a good example of synergis-
tic interaction of the two. Studies have shown that auxin
increases the expression of a cdc2 class of cyclin-de-
pendent kinases in tobacco pith explants. While the ex-
pression of the cdc2 like kinase is induced in response to
auxin, its catalytic activity is increased only when the
explants are also treated with cytokinin [15]. Cyclin δ 3
is a D cyclin whose expression is highly dependent on
cytokinin [16]. It is believed that δ 3 may be the factor
required to activate the cdc2 kinase. Therefore, the auxin
and cytokinin synergistically control the expression and
activity of the cdc2 like kinase, which renders the cell
competent for cell division (review, [1]).
Kakani et al. [17] recently found that exogenous cyto-
kinin can induce tissue specific up and down regulation

ARR5 and ARR6 Mediate Tissue Specific Cross-talk between Auxin and Cytokinin in Arabidopsis
550
of auxin in Arabidopsis. While auxin levels are reduced
by exogenous cytokinin in cotyledons, auxin is elevated
by exogenous cytokinin in roots and calli. More interest-
ingly, they found that AUX1 plays a critical role in medi-
ating cytokinin stimulated auxin accumulation in young
roots and calli. Müller and Sheen reported that cytokinin
and auxin interact antagonistically in root stem-cell
specification in early embryogenesis [18]. Auxin antago-
nizes cytokinin signaling by directly activating the rep-
ressors of cytokinin signaling, ARR7 and ARR15. How-
ever, how cytokinin and auxin interact during shoot stem
cell specification was not reported.
Both the auxin and cytokinin signaling pathways in-
volve highly complicated networks that contain a large
number of genes. The cytokinin signaling is perceived
via a phosphorelay that is similar to the two-component
systems used by bacteria for sensing and responding to
environmental stimuli (recent review [18,19]). The path-
way involves hybrid histidine protein kinases (AHK2,
AHK3 and CRE1/WOL/AHK4) as receptors, histine
phophotransfer proteins (AHPs), and nuclear response
regulators (type A-ARRs and type B-ARRs). There are
ten type-A ARR proteins (ARR3-ARR9 and ARR15-
ARR17) and 11 type-B ARR proteins (ARR1, ARR2,
ARR10-ARR14 and ARR18-ARR21) in Arabidopsis.
The type-A ARRs were originally identified as cytokinin
induced genes (review, [19]). At least eight of the ten
type-A ARRs are negative regulators of cytokinin sig-
naling with overlapping function. Both gene redundancy
and tissue-specific roles have been observed among type-
A ARRs in cytokinin response [20-22]. ARR5 and ARR6
share highest homology with each other compared with
other members in the same gene family [21]. ARR5 ex-
pression was found in the root and shoot meristems in the
absence of exogenous cytokinin [23]. In the presence of
exogenous cytokinin, the ARR5:GUS expression region
was enlarged to include tissues around the shoot meris-
tematic region in the shoot and all tissues in the roots,
from the hypocotyl-root junction through the root tip.
ARR6:GUS expression was detected in the shoot meris-
tematic region and cotyledon vasculature in young seed-
lings [21]. Cytokinin treatment resulted in overall higher
level expression of ARR6:GUS, including tissues in hy-
pocotyl and root except the root tip [21]. The arr5 knock-
out mutant has an altered rosette morphology, which dis-
appeared in arr5 arr6 double mutant, indicating that ARR5
and ARR6 have opposite functions [21].
Although the essential role of the interplay between
auxin and cytokinin in plant growth and development has
been well documented, little is known of the underlying
molecular mechanisms. Most of the reported studies lim-
ited to how the auxin and cytokinin levels modulated
each other and in meristem tissue development. In this
study, we report that the cytokinin induction of ARR5
and ARR6 expression is subjected to the regulation of
auxin in a tissue and gene specific manner. The distinct
auxin regulatory patterns of these two highly homolo-
gous genes provide novel insights into the mechanisms
underlying auxin and cytokinin interactions, especially
the tissue specific interactions of these two hormones.
2. Materials and Methods
2.1. Arabidopsis Growth and Callus Induction
Arabidopsis seeds were sterilized and placed on Gam-
borg’s B5 medium (pH 5.7) supplemented with 1% su-
crose, 0.9% agar, and 2,4-D (2,4-Dichlorophenoxyacetic
acid) and KT (Kinetin) as indicated in the text. After 5
days of cold treatment at 4˚C, the Petri dishes were
transferred to a 22˚C growth chamber for seed germina-
tion or callus induction under 16 hrs of light and 8 hrs of
dark. The light intensity was 150 mol m–2sec–2 for ger-
mination and 50 mol m–2sec–2 for callus induction.
2.2. ARR5:GUS and ARR6:GUS Transgenic
Lines
The Arabidopsis (Columbia) seeds of ARR5:GUS and
ARR6:GUS lines were ordered from Arabidopsis Bio-
logical Research Center (ABRC) at the Ohio State Uni-
versity [21].
2.3. Histochemical Analyses of GUS Activities
The histochemical stain of GUS was carried out as re-
ported by Sessions et al. [24] without sectioning. Briefly,
plant materials were stained in GUS staining solution
(100 mM Sodium Phosphate at pH 7.0, 10 mM EDTA,
0.1% Triton X-100, 1 mM potassium ferricyanide, 1 mM
potassium ferrocyanide and 1 mg/mL of X-Gluc [Gold
Bio Technology, Inc.]). The samples were incubated at
37˚C overnight after being placed under a vacuum for 10
min in a desiccator. The staining solution was removed
and the tissues were cleaned by incubating with several
changes of 70% ethanol. The GUS images are acquired
using a Zeiss Stemi SV11 (Apo) light Microscope.
3. Results and Discussions
3.1. Auxin Modulates ARR5 and ARR6
Expression with High Tissue Specificity and
a Contrast Pattern
In our previous studies, we found that cytokinin can in-
duce auxin redistribution in seedlings and calli and
AUX1 plays an essential role in mediating cytokinin in-
duced auxin redistribution [17]. To examine how the
cytokinin signaling pathways interact with auxin, we
examined the expression of cytokinin pathway genes in
response to exogenously applied auxin. ARR5 and ARR6

ARR5 and ARR6 Mediate Tissue Specific Cross-talk between Auxin and Cytokinin in Arabidopsis 551
share highest sequence homology in the Arabidopsis ge-
nome but may have opposite functions. We found that
ARR5 expression was in the root and shoot meristems of
wild-type seedlings as reported [23] (Figure 1(a)). In the
presence of 0.2 mg/l KT, the ARR5:GUS expression was
highly induced in roots, from the hypocotyl-root junction
through the root tip, and regions surrounding the shoot
apical meristem (Figure 1(b)). When auxin (2,4-D) was
also applied, the ARR5:GUS expression in roots were
strongly enhanced from the hypocotyl-root junction to
root tip (Figure 1(c)-(f)). In contrast, ARR5:GUS ex-
pression in cotyledons, shoot apical meristem, and hy-
pocotyls were suppressed.
When auxin concentration increased to 2.0 mg/l, the
ARR5:GUS expression was completely shut off in coty-
ledons and shoot apical meristem regions. But the gene
expression in roots was very high.
Interestingly, auxin regulation of ARR6 expression
followed a contrast pattern. In wild-type seedlings, ARR6:
GUS expression was detected in the shoot apical meris-
tematic region and cotyledon vasculature (Figure 2(a))
[21]. Cytokinin treatment elevated the expression of
ARR6:GUS, particularly in hypocotyls and roots as re-
ported (Figure 2(b)). When low auxin (0.05 mg/l and
0.25 mg/l 2,4-D) was applied, ARR6:GUS expression
was strongly induced in cotyledons, shoot apical meris-
tem region, and hypocotyls. On the other hand, its ex-
pression in roots was suppressed starting from the elon-
gation zone. With the increase of 2,4-D, the suppressed
region extended to hypocotyl region and root tip in both
directions. When 2,4-D reached 2.0 mg/l, ARR6:GUS
Figure 1. Histochemical assay of ARR5:GUS expression in
response to cytokinin and auxin induction. The seedlings
were germinated in B5 media supplemente d with KT and 2,
4-D in concentrations as indicated below. The seedlings
were five days old. (a) Wild-type seedling; (b) Seedling
grown in medium with 0.2 mg/l KT; (c) Seedling grown in
medium with 0.2 mg/l KT and 0.05 mg/l 2,4-D. (d) Seedling
grown in medium with 0.2 mg/l KT and 0.25 mg/l 2,4-D; (e)
Seedling grown in medium with 0.2 mg/l KT and 1.0 mg/l 2,
4-D; (f) Seedling grown in medium with 0.2 mg/l KT and 2.0
mg/l 2,4-D.
Figure 2. Histochemical assay of ARR6:GUS expression in
response to cytokinin and auxin induction. The seedlings
were germinated in B5 media supplemente d with KT and 2,
4-D in concentrations as indicated below. The seedlings
were five days old. (a) Wild-type seedling; (b) Seedling
grown in medium with 0.2 mg/l KT; (c) Seedling grown in
medium with 0.2 mg/l KT and 0.05 mg/l 2,4-D. (d) Seedling
grown in medium with 0.2 mg/l KT and 0.25 mg/l 2,4-D; (e)
Seedling grown in medium with 0.2 mg/l KT and 1.0 mg/l 2,
4-D; (f) Seedling grown in medium with 0.2 mg/l KT and 2.0
mg/l 2,4-D.
was expressed only in cotyledons. The expression in
other tissues was almost completely suppressed. When
the ARR6:GUS and ARR5:GUS expression patterns
were compared (compare Figure 1 with Figure 2), it was
clear that the expression of ARR5:GUS and ARR6:GUS
followed a reversed pattern with the increase of auxin,
suggesting a tissue specific role of these two genes in the
interplay between auxin and cytokinin.
While examining the mutant phenotype in Arabidopsis
[21], it was observed that the reduced rosette size of ARR5
mutant was not enhanced by the mutation of ARR6. In-
stead, the phenotype disappeared in the double mutant,
indicating a different function of these two genes al-
though these two genes have a high level of spatial over-
lapping in gene expression in cotyledons and hypocotyls
in wildtype seedlings. Given that ARR5 and ARR6 nega-
tively regulate cytokinin response, our observations sug-
gest that auxin suppress the cytokinin stimuli in plants via
modulation of the negative regulators in the cytokinin
pathway. The tissue specificity of auxin and cytokinin
cross-talk is achieved via differential regulation of the
highly homologous genes with different functions.
3.2. Auxin Regulation of ARR5 and ARR6
Expression in Calli
To understand auxin and cytokinin interaction in calli,
we examined the expression of ARR5:GUS and ARR6:
GUS in calli induced from germinating seeds with dif-
ferent auxin/cytokinin ratios and culturing time. ARR5:
GUS was highly expressed in calli (Figure 3). Some
cells, derived from cotyledons, barely expressed ARR5

ARR5 and ARR6 Mediate Tissue Specific Cross-talk between Auxin and Cytokinin in Arabidopsis
552
Figure 3. Histochemical assay of ARR5:GUS expression in
calli. The calli were induced in B5 media supplemented with
KT and 2.4-D in concentrations as indicated below. (a) Four
weeks old calli induced by 0.2 mg/l KT and 0.05 mg/l 2.4-D;
(b) Four weeks old calli induced by 0.2 mg/l KT and 0.25
mg/l 2.4-D; (c) Four weeks old calli induced by 0.2 mg/l KT
and 1.0 mg/l 2.4-D; (d) Four weeks old calli induced by 0.2
mg/l KT and 2.0 mg/l 2.4-D; (e) Six weeks old calli induced
by 0.2 mg/l KT and 2.0 mg/l 2.4-D; (f) Eight weeks old calli
induced by 0.2 mg/l KT and 2.0 mg/l 2.4-D.
initially when auxin was high. ARR5:GUS became
highly expressed across the entire callus after eight
weeks of culture. In contrast, the expression of ARR6:
GUS in calli was low (Figure 4). Although small portion
of the tissues, which derived from cotyledons, expressed
ARR6:GUS initially, the GUS expression substantially
decreased with time. After eight weeks, the ARR6:GUS
expression was barely detected in the calli. Our results
suggest that ARR5 is highly expressed in calli but ARR6
is silenced in calli.
Figure 4. Histochemical assay of ARR6:GUS expression in
calli. The calli were induced in B5 media supplemented with
KT and 2.4-D in concentrations as indicated below. (a) Four
weeks old calli induced by 0.2 mg/l KT and 0.05 mg/l 2.4-D;
(b) Four weeks old calli induced by 0.2 mg/l KT and 0.25
mg/l 2.4-D; (c) Four weeks old calli induced by 0.2 mg/l KT
and 1.0 mg/l 2.4-D; (d) Four weeks old calli induced by 0.2
mg/l KT and 2.0 mg/l 2.4-D; (e) Six weeks old calli induced
by 0.2 mg/l KT and 2.0 mg/l 2.4-D; (f) Eight weeks old calli
induced by 0.2 mg/l KT and 2.0 mg/l 2.4-D.
4. Conclusions
The interaction of auxin and cytokinin is essential to
plant growth and development. The molecular mecha-
nisms underlying the interaction, particularly the tissue
specific response, are still poorly understood. Our results
reported here suggest that auxin interacts with the cyto-
kinin pathway by directly regulating the expression of
negative regulators ARR5 and ARR6 in the cytokinin
signaling pathway. The tissue specific response is a-
chieved by differential regulation of homologous genes
with distinct functions.
5. Acknowledgements
We are grateful to Dr. Scott Willard, Dr. Jiaxu Li, Dr.
Din-Pow Ma, and Ms Hana Mujahid for a critical reading
of this manuscript. This research was approved for pub-
lication as Journal Article J-10944 of the MAFES, Mis-
sissippi State University.
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