Plant Cell Physiol.

48(1): 84–96 (2007)

doi:10.1093/pcp/pcl040, available online at www.pcp.oxfordjournals.org
ß The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Type-B ARR Transcription Factors, ARR10 and ARR12, are Implicated

in Cytokinin-Mediated Regulation of Protoxylem Differentiation in
Roots of Arabidopsis thaliana
Akihiro Yokoyama 1, Takafumi Yamashino 1, *, Yu-Ichiro Amano 1, Yoshinori Tajima 1, Aya Imamura 1,
Hitoshi Sakakibara 2 and Takeshi Mizuno 1
1
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Furocho, Chikusa, Nagoya, 464-8601 Japan
2
RIKEN Plant Science Center, 1-7-22, Suehiro, Tsurumi, Yokohama, 230-0045 Japan

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In the phosphorelay-mediated cytokinin signal
transduction of Arabidopsis thaliana, certain members of
the type-B authentic response regulator (ARR) family are
implicated in the regulatory networks that are primarily
propagated by the cytokinin-receptors [authentic histidine
kinases (AHKs)]. Clarification of the involvement of each
type-B ARR transcription factor in cytokinin-responsive
phenomena is still at a very early stage. Here we analyzed
the redundant function of two type-B ARR genes, ARR10
and ARR12, by constructing an arr10/arr12 double mutant.
The resulting mutant plants showed stronger phenotypes
with special reference to the cytokinin action in roots
(e.g. inhibition of root elongation, green callus formation
from root explants) than those for each single mutant,
suggesting that ARR10 and ARR12 redundantly play an
important role in the cytokinin signaling in roots. This idea
was further supported by results from root-specific micro-
array analyses with the double mutant plant. We also showed
that ARR10 and ARR12 are involved in the AHK-dependent
signaling pathway that negatively regulates protoxylem
specification in root vascular tissues. When the double
mutant is combined with an arr1 allele, the resultant
arr1/arr10/arr12 triple mutant showed phenotypes displaying
a very poor growth, quite similar to those of the wooden
leg (wol) mutant that virtually lacks cytokinin receptor
activities in plants. In this triple arr mutant, the specification
of root vascular tissues is also affected as severely as in wol.
Taken together, we propose that ARR10 and ARR12,
together with ARR1, redundantly play pivotal roles in the
AHK-dependent phosphorelay signaling in response to
cytokinin in roots.
Keywords: Arabidopsis thaliana — ARR — Cytokinin —
Microarray — Phosphorelay — Root differentiation.
Abbreviations: ACS, 1-aminocyclopropane-1-carboxylate
synthase; AHK, authentic histidine kinase; AHP, authentic
histidine-containing phosphotransmitter; ARR, authentic
response regulator; AtHK1, Arabidopsis histidine kinase 1;
BA, 6-benzylaminopurine; Col, Columbia-0; DMSO, dimethyl-
sulfoxide; FC, fold change; GUS, b-glucuronidase; RT–PCR,
reverse transcription–PCR; wol, wooden leg.
Introduction
Cytokinins are a class of plant hormones which are
implicated in nearly all aspects of plant growth and
development, including cell division, shoot initiation and
light responses (Mok and Mok 2001). In Arabidopsis
thaliana, the immediate early response of plants to cytokinin
has been formulated as the multistep authentic histidine
kinase (AHK)–authentic histidine-containing phospho-
transmitter (AHP)–authentic response regulator (ARR)
phosphorelay signaling, which is initiated by the cytokinin
receptor histidine protein kinases (AHK2, AHK3 and
AKH4/CRE1/WOL) (for recent reviews, see Hwang et al.
2002, Schaller et al. 2002, Kakimoto 2003, Heyl and
Schmülling 2003, Mizuno 2004, Ferreira and Kieber
2005). These cytokinin receptor histidine kinases sense the
signal, and phosphorylate histidine-containing phospho-
transfer intermediates (AHPs) (Inoue et al. 2001, Suzuki
et al. 2001, Ueguchi et al. 2001a, Ueguchi et al. 2001b,
Yamada et al. 2001). The phosphorylated AHPs move into
the nucleus and donate the phosphoryl group to type-B
ARRs containing a phospho-accepting aspartate residue
(Suzuki et al. 1998, Hwang and Sheen 2001, Imamura
et al. 2001, Suzuki et al. 2002, Tanaka et al. 2004, Yamada
et al. 2004). The phosphorylated type-B ARRs serve as
transcriptional activators, resulting in rapid induction of
cytokinin-associated target genes (Sakai et al. 2000, Sakai
et al. 2001, Hosoda et al. 2002, Imamura et al. 2003),
including a set of type-A ARR genes (Brandstatter and
Kieber 1998, Kiba et al. 1999, D’Agostino et al. 2000, Sakai
et al. 2001). Accumulated type-A ARRs somehow act
as negative regulators in the signaling of cytokinin (Hwang
and Sheen 2001, Kiba et al. 2003, To et al. 2004, Leibfried
et al. 2005). To support this scenario, several lines of
forward and reverse genetic evidence have currently
been accumulating (Mähönen et al. 2000, Kiba et al.
2002, Osakabe et al. 2002, Suzuki et al. 2002, Imamura
et al. 2003, Kiba et al. 2004, Mason et al. 2004, Tajima et al.
2004). For instance, the mutant plants lacking all three
cytokinin receptors were constructed, demonstrating that

*Corresponding author: E-mail, yamasino@agr.nagoya-u.ac.jp; Fax, þ81-52-789-4091.

84
 Cytokinin responses in Arabidopsis thaliana
such cytokinin receptor-less (or cytokinin-insensitive) plants
are viable, but very dwarf and sterile, implying that the
AHK-mediated phosphorelay circuitry plays pivotal
roles for plant development in total (Higuchi et al. 2004,
Nishimura et al. 2004, Kim et al. 2006, Riefler et al. 2006).
The scenario described above has been extensively
documented and discussed in many recent review articles,
emphasizing that the AHK2/3/4-dependent histidine–
aspartate phosphorelay is central to the biological actions
of cytokinins in plants (Hwang et al. 2002, Schaller et al.
2002, Kakimoto 2003, Heyl and Schmülling 2003, Mizuno
2004, Ferreira and Kieber 2005). Nevertheless, this current
scenario does not necessarily describe the whole picture of
the cytokinin-responsive histidine–aspartate phosphorelay
signaling in A. thaliana, because anonymous AHK/AHP/
ARR players appear on the scene (for recent reviews, see
Grefen and Harter 2004, Mizuno 2004, Mizuno 2005).
In addition to three AHK cytokinin receptors, in fact, this
model plant has five members of AHPs, 11 members of
type-B ARRs and 10 members of type-A ARRs (Imamura
et al. 1998, Imamura et al. 1999, Mason et al. 2004, Tajima
et al. 2004, To et al. 2004). The critical question is then:
where, when and how do these AHKs/AHPs/ARRs play
their roles in plants with which kind of combinations
during what type of cytokinin-mediated biological events?
As an approach to this end, here we studied the type-B
ARR family of transcriptional regulators in the hope that
they would provide us with new insight into the issues
addressed above.
Obviously, a direct approach to define the physio-
logical function of a given type-B ARR transcription factor
would be to characterize each loss-of-function (or null)
mutant. A transfer (T)-DNA insertion mutant of a
representative type-B ARR gene (ARR1) was the first to
be characterized (Sakai et al. 2001). The arr1 mutant plants
were slightly insensitive to cytokinin in the inhibition of
root elongation; otherwise they grew up and set seeds
as well as the wild-type plants. This subtle effect of the
arr1 loss-of-function mutant can be explained by assuming
that at least one other type-B ARR member plays a
partially redundant role. Thus, further combinatorial
genetic analyses are needed to complement the character-
ization of each single mutant that may not display an overt
phenotype. It was recently demonstrated that such higher
order mutants, including an arr1 arr10 arr12 triple mutant,
display progressively increasing resistance to cytokinin
in the root elongation assay, but the overall phenotypes
observed for the triple mutant were not as severe as those
of the ahk2 ahk3 ahk4 triple mutant (Mason et al. 2005).
To extend this type of forward genetics, we characterized
a newly established arr10 arr12 double null or strong
hypomorphic mutant, together with a new arr1 arr10 arr12
triple mutant. In this study, we show that these mutants
85
display strong phenotypes that are extremely relevant to
the action of cytokinin in roots, providing us with new
insight into the biological impact of histidine–aspartate
phosphorelay signaling in A. thaliana
Results and Discussion
A phylogenetic overview of type-B ARRs in Arabidopsis
and rice
The Arabidopsis type-B ARR family consists of 11
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members (see those denoted by red letters in Fig. 1). To gain
an idea about the functional differentiation of type-B ARR
genes, here we incorporated a set of homologous type-B RR
sequences from rice (Oryza sativa) into a comparative
phylogenetic tree (see those denoted by blue letters
in Fig. 1). It is currently believed that seven of the ARR
members (ARR1, 2, 10, 11, 12, 14 and 18) are implicated
in cytokinin signaling (Sakai et al. 2000, Imamura et al.
2003, Mason et al. 2004, Mason et al. 2005).
Others (ARR13, 19, 20 and 21) are distantly related to
these cytokinin-associated ARRs, and their functions have
not yet been clarified (Tajima et al. 2004). In this respect,
the analysis with the rice RR sequences provided us with
several interesting views, which are consistent with those
noted previously (Doi et al. 2004, Ito and Kurata 2006,
Pareek et al. 2006). This model monocot has at least seven
genes, each of which encodes a putative type-B RR. One of
them is Ehd1, which has been implicated in the control
of flowering time in rice (Doi et al. 2004). It is clear from
the phylogenetic tree that the remaining six rice RRs
are all closely related to the cytokinin-associated ARRs.
Furthermore, each of these rice RRs has its possible
counterpart in Arabidopsis. In particular, the pair ARR10
and ARR12 is highly homologous to a set of rice RRs
(Os02g0182100, Os02g0796500 and Os06g0183100),
while the best characterized ARR1 of A. thaliana has no
apparent counterpart in rice. Based on the results from
such comparative analyses, we considered a priori that the
pair ARR10 and ARR12 may be the promising genes of
choice for further extensive genetic studies.
Expression profiles of ARR10 and ARR12 in plants
The tissue- or organ-specific expression profiles of the
type-B ARR family of genes were previously examined to
some extent, suggesting that the transcripts of ARR10 and
ARR12 are detected in both leaves and roots, and at almost
every developmental stage examined (Mason et al. 2004,
Tajima et al. 2004). To gain further insight into ARR10 and
ARR12, here we re-examined the expression profiles
of ARR10::GUS and ARR12::GUS by employing a set of
independent transgenic lines. Such representative results
are shown in Fig. 2 (7-day-old seedlings). The expression
of both ARR10 and ARR12 was predominant in
86 Cytokinin responses in Arabidopsis thaliana

ARR1 ARR10::GUS ARR12::GUS

ARR2
ARR11
Os01g0904700
ARR10

Cytokinin Responses
ARR12
Os02g0796500
Os06g0183100
Os02g0182100
ARR18
Os06g0647200

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Os03g0224200
ARR14 a d
Flowering
Os10g0463400 (Ehd1)
ARR19
Unknown

ARR20
ARR13
ARR21

Fig. 1 A phylogenetic tree of the type-B RR family of response

regulators from A. thaliana and O. sativa. Arabidopsis thaliana has
11 type-B ARR members, as is well known (indicated by red
letters). An inspection of the current rice (O. sativa) genome
databases revealed at least seven homologous type-B RRs
(indicated by blue letters). For this inspection, we mainly used
the database from the Rice Annotation Project (Ohyanagi et al.
2005, http://rapdb.lab.nig.ac.jp/index.html). The nomenclature of b e
each inferred rice RR gene was also adopted from the database.
Recent reports on the rice RR genes were also referred to (Doi et al.
2006, Ito et al. 2006, Pareek et al. 2006). Using the amino acid
sequences of receiver domains from these 18 type-B RRs from
A. thaliana and O. sativa, a non-rooted neighbor-joining
phylogenetic tree was constructed by the program Clustal W.

a meristematic region in apical parts (Fig. 2a, d), while the

cotyledons were strongly stained in ARR12 but not in
ARR10. The expression of ARR10 and ARR12 was also
evident in roots (Fig. 2b, c, e, f). However, their expression
profiles were characteristic for each; the expression of
ARR10 was predominantly observed at both the primary
and lateral root tips where cells are vigorously dividing
(Fig. 2b, c), whereas the expression of ARR12 appeared
to be confined to the vasculature tissues (Fig. 2e, f).
These results were mainly consistent with those reported
previously (Mason et al. 2004, Tajima et al. 2004).
However, some differences from the previous results were
observed in the staining profiles of lateral roots. This may
be due to the differences in the developmental stages
examined. In any case, it may be noted that the expression
profiles of ARR10 and ARR12 in roots are complementary
in part to each other. Keeping this observation in mind, an
arr10 arr12 double mutant was isolated to characterize the
actions of cytokinin in roots.
Isolation of T-DNA mutant lines
For ARR10 and ARR12, a set of mutant lines
was established by employing the candidate resources of
c f
Fig. 2 Expression patterns of the GUS reporter gene under control
of regulatory sequences from the ARR10 or ARR12 gene.
Each promoter::GUS fusion contained an approximately 1.5 kb
DNA fragment upstream from its own predicted start codon,
which would include a potential promoter (Tajima et al. 2004).
Seedlings (7-day-old) carrying either ARR10::GUS (a, b or c)
or ARR12::GUS (d, e or f) were histochemically analyzed.
To improve data consistency, such histochemical analyses were
conducted on several independently grown sets of plants of at
least two independent transgenic lines. Those represented are
typical staining profiles: aerial parts (a, d), developing lateral roots
(b, e) and primary root tips.
T-DNA insertion lines from the SALK Institute
(Alonso et al. 2003) through the Arabidopsis Biological
Resource Center (Columbus, OH, USA). Those established
here are the homozygous lines, designated as arr10-5 and
arr12-1, respectively (Fig. 3A). As judged by the analyses of
 Cytokinin responses in Arabidopsis thaliana
500bp arr10-5
A arr10-2
a b c arr10
Receiver
arr12-1 GARP
d e
B Col arr10-5
a/c Col arr12-1
a/b d/e
ACT8 ACT8
Fig. 3 Identification of the ARR10 and ARR12 mutant alleles.
(A) Location of the T-DNA insertion points in the ARR10 and
ARR12 coding sequences on the genome. The ARR10 and ARR12
coding sequences are schematically shown (rectangles, exons;
horizontal lines, intron). The regions corresponding to receiver
domains and GARP DNA-binding motifs are also indicated.
The identified T-DNA insertion sites are indicated, and the resultant
mutant alleles were designated as arr10-5 and arr12-1, respec-
tively. The arr10-2 allele has been identified previously
(Mason et al. 2005). (B) RT–PCR analyses of transcripts. mRNA
samples were prepared from wild-type (Col), arr10-5 and arr12-1
plants, and they were analyzed by RT–PCR with the appropriate
pair of primers (see arrows denoted by a, b, c, d, e in A, and
see also Materials and Methods for details).
ARR10 and ARR12 transcripts in the respective mutant
plants, both of the arr mutant alleles are assumed to be null
or hypomorphic (Fig. 3B). Through the subsequent genetic
cross with these, an arr10-5 and arr12-1 double mutant was
also isolated (hereafter designated ‘arr10/12’ for clarity).
These were derivatives of the ecotype Columbia-0 (Col).
It may be worth mentioning that the arr12-1 allele is
the same as that isolated previously by another group
(Mason et al. 2005). However, arr10-5 is different from the
allele isolated previously, which has a T-DNA insertion
at the very 30 end of ARR10 (see Fig. 3A, arr10-2)
(Mason et al. 2005). These homozygous mutant plants
(arr10, arr12, and arr10/12) grew well on both MS agar
plates and soil, and then they set seeds as normally as the
wild-type Col. We thus examined closely the phenotypes of
these type-B arr mutants with special reference to the
cytokinin action in roots.
Phenotypes with regard to the inhibition of root
elongation by cytokinin
A hallmark cytokinin action on roots is its inhibitory
effect on root elongation (Cary et al. 1995). The mutants,
arr10, arr12 and arr10/12 were grown on vertically
orientated MS agar plates containing various concentra-
tions of a cytokinin [6-benzylaminopurine (BA)] (Fig. 4A),
87
A
Col 5 mm
arr12
arr10/12
BA (10 µM)
B 60
Col
arr10
arr12
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50
arr10/12
40
Root length (mm)
30
20
10
0
0 0.001 0.01 0.1 1 10
BA (µM)
Fig. 4 Phenotypes of a set of arr mutants with regard to
the inhibition of root elongation by cytokinin. (A) Photographic
representation of the root lengths of young seedlings (8-day-old),
which were grown under continuous light at 228C on vertically
oriented MS agar plates supplemented with 10 mM BA.
Representative seedlings of each arr mutant, together with Col,
were photographed and are shown. (B) Dose response for the effect
of cytokinin on the set of arr mutants. Seedlings were grown on
MS agar plates supplemented with the indicated concentrations
of BA under the same conditions as those described for (A).
The lengths of roots of young seedlings (10-day-old) were
measured and are presented with error bars (SD from n410).
and the lengths of the resulting primary roots were
measured (Fig. 4B). The results showed that the roots of
arr12 were slightly less sensitive to cytokine in this assay,
while the response of arr10 was indistinguishable from that
of the wild-type roots. When these weak mutant alleles were
combined together, the roots of arr10/12 were markedly less
sensitive to cytokinin in this assay. This event was best
explained by assuming that ARR10 and ARR12 play a
cytokinin-associated role(s) redundantly in roots. However,
the results also suggest that their roles are not completely
redundant because the arr12 single mutant also showed the
phenotype, but the arr10 single mutant did not. This latter
notion is consistent with the results showing that
their expression profiles are distinct from each other in
roots (see Fig. 2).
88 Cytokinin responses in Arabidopsis thaliana
t-zeatin (µM)
0.005 0.05 0.5 5 25 0.005 0.05 0.5
0.5
2,4-D (µM)
0.05
Col arr12
0.5
0.05 arr10 arr10/12
Fig. 5 Phenotypes of a set of arr mutants with regard to in vitro
callus formation. With the set of arr mutants, seedlings were grown
on MS agar plates for a week. The primary roots were excised,
and the pieces of explants were transferred onto MS agar plates
supplemented with various concentrations of t-zeatin and 2,4-D.
The plates were incubated for 43 d under continuous light at 228C.
From each plate, representative calli were collected and
appropriately arranged, as shown.
Phenotype with regard to the cytokinin-dependent green
callus formation from explants
Another hallmark cytokinin action on root tissues is
the formation of a green callus from explants (Mok and
Mok 2001). The segments of primary roots were detached
from the set of mutants, and these explants were placed on
MS agar plates containing cytokinin (trans-zeatin) and
auxin (2,4-D) in various concentrations, as indicated in
Fig. 5. As is well known, when the concentrations of both
t-zeatin and 2,4-D were optimized relative to each other,
green calli with shoots were vigorously developed from the
root explants (Fig. 5). It is of note that the explants from
arr10/12 failed to do this, while those from the arr10 and
arr12 single mutants responded as well as in the case of the
wild-type explants. These results further supported the
above view that ARR10 and ARR12 are involved in
cytokinin-mediated signal transduction in roots, in which
they play a redundant (and/or complementary) role(s).
As an appropriate reference, we also employed an arr1 null
mutant (this allele was designated as arr1-4, see below and
Mason et al. 2005). As in the case of arr10/arr12, the root
explants from the arr1/arr12 double mutant failed to
develop green calli (data not shown). As judged by these
severe phenotypes of arr10/12 and arr1/arr12, it was
suggested that ARR10 and ARR12, together with ARR1,
appear to be the prominent players in cytokinin signaling in
roots, as far as the regulation of root elongation and in vitro
callus formation are concerned.
Root-specific microarray analyses with the arr10/12 double
mutant
To examine further the nature of the arr10/12 double
mutation at the level of transcription, we then adopted
microarray technology. We and others have already
reported several papers dealing with microarray analyses
with reference to cytokinin action and histidine–aspartate
5 25 phosphorelay signaling (Che et al. 2002, Hoth et al. 2003,
Rashotte et al. 2003, Kiba et al. 2004, Kiba et al. 2005).
In such analyses, however, RNA samples were mainly
prepared from leaves without roots. In the context of this
study, we analyzed the RNA samples prepared selectively
from the cytokinin-treated roots. This was done for RNA
samples (three independent preparations) from Col and
arr10/12, and the essential data are presented in Table 1 and
Fig. 6.
In Table 1, a set of cytokinin-responsive (up-regulated
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and down-regulated) genes in the wild-type roots is listed
[mean fold-change (FC) 42.5 and 50.4, respectively].
They were then compared with those from arr10/12
(see the column of FC in arr10/12). To obtain an overall
view of this complicated result, each normalized expression
level of the 36 genes up-regulated by t-zeatin in Col was
visualized by its color intensity, as depicted in Fig. 6A
(high expression, red; low expression, green). It was evident
that the induction levels of many cytokinin-inducible genes
in Col were markedly attenuated in arr10/12 (compare the
level of reddishness in the second and third columns).
This is consistent with the idea that ARR10 and ARR12
coordinately play significant roles in cytokinin-responsive
transcriptional regulation in roots. Note also that the entire
raw data sets have been deposited in the NASCArrays
(http://affymetrix.arabidopsis.info/donating.html) so as to
be publicly available [reference numbers: NASCARRAYS-
405 (aerial parts) and NASCARRAYS-406 (roots)].
Specific insight into the microarray analyses for arr10/12
These data for cytokinin-responsive genes in roots
were compared with those reported previously for leaves
(Kiba et al. 2005), and the genes that were commonly
up-regulated by t-zeatin in both roots and leaves were
indicated (see the genes denoted by asterisks in Table 1).
A considerably large number of genes (14 out of 36 genes)
were classified into this group. These included an AS2
family gene (At1g16530, ASL9) (Yang et al. 2006) and a
cytokinin oxidase family gene (At4g29740, CKX4)
(Schmülling et al. 2003). In addition, it was quite reasonable
that a set of type-A ARR family genes (ARR5, 6, 7, 15 and
16) was also included in this group.
This analysis further uncovered several other genes
which were induced by t-zeatin in a root-specific manner.
They included a cytochrome P450 family gene that was
previously implicated in the synthesis of t-zeatin
(At1g67110, CYP735A2) (Takei et al. 2004), one of the
Arabidopsis histidine kinase genes (At2g17820, AtHK1)
(Urao et al. 2001) and two expansin family genes
(At5g56320 and At2g03090, EXP14 and EXP15) (Lee
et al. 2001). The cytokinin-dependent induction of these
three genes was markedly attenuated in the arr10/12 double
 Cytokinin responses in Arabidopsis thaliana 89

Table 1 Root-specific microarray analyses of cytokinin-regulated genes of wild-type and arr10/12 plants
Products FC in Col FC in arr10/12
Induced genes
At2g40670
Response regulator (ARR16) 8.1 3.7
At1g67110 Cytochrome P450 (CYP735A2) 6.3 1.9
At2g30540 Glutaredoxin family protein 6.3 8.7
At1g19050
Response regulator (ARR7) 6.0 4.6
At5g14070 Glutaredoxin family protein 5.6 3.3
At1g74890
Response regulator (ARR15) 5.5 3.5
At3g48100
Response regulator (ARR5) 5.0 4.3

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At3g61880 Cytochrome P450 (CYP78A9) 4.1 2.0
At2g17820 Histidine kinase (AtHK1) 3.9 1.6
At3g29670
Transferase family protein 3.5 2.5
At1g16530
AS2 family protein (ASL9) 3.4 2.7
At5g62920
Response regulator (ARR6) 3.3 2.4
At5g11160 Adenine phosphoribosyltransferase 3.2 1.6
At4g29740
Cytokinin oxidase family protein (CKX4) 3.1 2.6
At4g39770
Trehalose-6-phosphate phosphatase 3.1 1.2
At5g56320 Expansin (EXP14) 3.1 1.3
At4g25410 Basic helix–loop–helix (bHLH) family protein 3.0 2.2
At1g58170
Disease resistance-responsive protein-related 2.9 1.4
At2g25160 Cytochrome P450 (CYP82F1) 2.9 2.0
At5g56970 Cytokinin oxidase family protein (CKX3) 2.9 1.8
At1g49450 WD-40 repeat family protein 2.9 2.6
At3g51680 Short-chain dehydrogenase/reductase (SDR) family protein 2.8 1.7
At3g45700 Proton-dependent oligopeptide transport (POT) family protein 2.8 1.9
At2g40230
Transferase family protein 2.8 1.4
At1g58340 MATE efflux protein-related 2.7 2.1
At4g02850
Phenazine biosynthesis PhzC/PhzF family protein 2.6 1.8
At3g50300 Transferase family protein 2.6 1.7
At4g15690
Glutaredoxin family protein 2.6 1.4
At2g32510 Protein kinase family protein 2.6 2.1
At3g57010
Strictosidine synthase family protein 2.5 1.2
At2g03090 Expansin (EXP15) 2.5 1.2
At1g68360 Zinc finger protein-related 2.5 1.7
(continued)

mutant (Table 1). In addition to these up-regulated genes,
several down-regulated genes might also be interesting. They
included several genes for transcription factors, together
with an adenosine phosphate-isopentenyltransferase gene
that plays a key role in the de novo production of cytokinins
(At3g23630, IPT7) (Kakimoto 2001, Takei et al. 2001,
Kieber et al. 2002). Although we did not characterize these
genes any further in this study, they will provide us with
hints for future studies with regard to the molecular linkages
between these genes and cytokinin signaling in roots.
Meanwhile, it may be noteworthy again that ARR5, 6,
7, 15 and 16 were reproducibly identified as the cytokinin-
inducible genes in the wild-type roots (Table 1). Therefore,
we examined these events in detail (Fig. 6B). The levels of
induction of ARR15 and ARR16 were more severely
attenuated in the arr10/12 roots, as compared with other
cases, i.e. ARR5, ARR6 and ARR7. To confirm this, we
prepared RNA samples from the roots of plants grown
under essentially the same conditions as the microarray
analyses, and they were subjected to semi-quantitative
reverse transcription–PCR (RT–PCR) analyses (Fig. 6C).
The results were essentially consistent with those of the
microarray analyses. Although these results do not mean
that ARR15 and ARR16 are the specific targets of the
ARR10 and ARR12 transcription factors, at least it is
tempting to speculate on the occurrence of a signaling
stream of AHKs–ARR10/12–ARR15/16 in roots
(see Fig. 9).
90 Cytokinin responses in Arabidopsis thaliana

Table 1 Continued
Products FC in Col FC in arr10/12
Repressed genes
At3g62550 Universal stress protein (USP) family protein 0.40 1.13
At5g07580 Ethylene-responsive element-binding family protein 0.40 0.66
At1g43160 AP2 domain-containing protein RAP2.6 (RAP2.6) 0.39 0.53
At4g33420 Peroxidase 0.39 0.79
At5g54030 DC1 domain-containing protein 0.38 0.79
At5g09440 Phosphate-responsive protein 0.36 0.61
At3g04530 Phosphoenolpyruvate carboxylase kinase 2 (PPCK2) 0.35 0.49

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At5g01210 Transferase family protein 0.35 0.60
At2g23170 Auxin-responsive GH3 family protein 0.34 0.46
At1g51820 Leucine-rich repeat protein kinase 0.33 0.94
At2g22880 VQ motif-containing protein 0.33 0.81
At3g48920 Myb family transcription factor (MYB45) 0.32 0.34
At3g23630 Adenosine phosphate-isopentenyltransferase (IPT7) 0.30 0.60
At5g53980 Homeobox-leucine zipper family protein 0.30 0.37
At2g43140 Basic helix–loop–helix (bHLH) family protein 0.29 0.56
At3g58190 AS2 family protein (ASL16) 0.24 0.39
At5g40590 DC1 domain-containing protein 0.19 0.31
Detailed procedures of microarray analyses are given in Materials and Methods. Briefly, to identify cytokinin-regulated genes, the raw
data were analyzed by using a comparative analysis algorithm in GeneSpring 7.3 software (Agilent Technologies). The mean fluctuation
ratios of the normalized transcript level were calculated for each gene, and they were expressed as fold change (FC). A total of 63 genes
whose expression was significantly increased or decreased in Col in response to cytokinin were selected and listed [mean FC 42.5
(36 genes) or50.4 (27 genes)]. Asterisks indicate the up-regulated genes whose expression was also up-regulated by cytokinin in leaves in a
previous study (Kiba et al. 2005). The results for arr10/12 are also listed. Note that the genes encoding ‘anonymous expressed proteins’
were excluded from this table for clarity. They include four up-regulated genes (At1g13740, At3g05770, At3g46880 and At5g22390) and 10
down-regulated genes (At1g21360, At1g79160, At2g28305, At2g39370, At2g40435, At2g41230, At2g43060, At3g47510, At4g25760 and
At5g64890).

It should finally be noted that recently Rashotte et al.
(2006) reported the data set of microarray analysis with an
arr1 arr12 double mutant. Their experimental conditions
(e.g. growth stage, cytokinin treatment and oligonucleotide
microarray chip) were considerably different from ours. It is
rather difficult to compare their results directly with ours.
However, the microarray data sets of both the arr1 arr12
and arr10 arr12 double mutants will provide us with
valuable information.
ARR10 and ARR12 are involved in cytokinin-mediated
regulation of cell fate during root vascular development
Recently it was reported that cytokinin regulates cell
fate during root vascular development (Mähönen et al.
2006a). More specifically, cytokinin negatively regulates
the differentiation ‘from procambium to protoxylem’
(or protoxylem specification) (Fig. 7A). The AHK-
dependent cytokinin signaling pathway is critical in this
proposed model. If, as we proposed, ARR10 and ARR12
were crucial in the AHK-dependent cytokinin signaling
pathway in roots, then the arr10/arr12 double mutant
should display a clear phenotype with respect to this
particular event. We addressed this critical issue, and the
results showed that this is the case (Fig. 7B, C).
In this experiment, we employed a reference mutant,
ahk3-3/cre1-12, which lacks two of the cytokinin-receptor
genes (AHK3 and CRE1/AHK4/WOL) (Higuchi et al.
2004). In addition to this appropriate control (designated as
ahk3/4), Col and arr10/12 were grown on MS plates in the
presence and absence of BA (see 4-day-old seedlings in
Fig. 7B, note also that not only arr10/12, but also ahk3/4
was resistant to BA in the inhibition of root elongation).
They were then stained with fuchsin to visualize protoxylem
under a confocal microscope. The regions where fluorescent
images were observed are approximately indicated by red
arrows in Fig. 7B. As reported previously, when the roots of
Col were treated with a low concentration (0.1 mM) of BA,
the differentiation from procambium to protoxytem was
inhibited (Fig. 7C, see yellow arrows). In contrast, such an
inhibitory effect of BA was not observed for the roots of
arr10/12, even when they were treated with a very high
concentration (10 mM) of BA. Likewise, the roots of ahk3/4
did not show any response to BA under the conditions
tested. Rather, a few extra protoxylem files were seen in
the roots of ahk3/4, suggesting that the protoxylem
 Cytokinin responses in Arabidopsis thaliana 91

A Col arr10/12 specification was accelerated in this particular mutant

t-Zeatin
(see extra yellow arrows in Fig. 7C). This observation is
− + + −
FC=8 consistent with the model proposed by Mähönen et al.
(2006a) (Fig. 7A). Taken together, it was suggested that
ARR10 and ARR12 are the crucial transcription factors
36 Induced-Genes in Col

that act downstream of AHK3 and AHK4, both of which

perceive a cytokinin signal and consequently regulate cell
fate during root vascular development.
For these experiments, the arr10/12 mutant roots were
treated with externally added cytokinin. Then, we carefully
examined the development of root vascular tissues of

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arr10/12, which were grown in the absence of BA
FC=2.5 (Fig. 7C, and data not shown). Nevertheless, no character-
Expression Level
Low High istic anomaly was detected for arr10/12 under these
−0.25 −0.5 1 2 4
conditions, suggesting that the double lesions of arr10/12
B * are rather subtle in the absence of external cytokinin.
Col
8 arr10/12 This suggested that although ARR10 and ARR12 coordi-
Relative Amount of Transcripts(FC)

7 nately play certain roles in root vascular development, there

6 * must be an additional player(s) that acts redundantly with
5 ARR10 and ARR12, as further addressed below.
4 *
Characterization of an arr1/10/12 triple mutant with regard
3
to root vascular development
2
To address the above issue, we finally established
1 a homozygous arr1-4/arr10-5/arr12-1 triple mutant.
1/
2 The resulting arr1/10/12 triple mutant plants reproducibly
ARR15
ARR16
ARR17
AtHK1
AHK2
AHK3
AHK4
AHK5

ARR3
ARR4
ARR5
ARR6
ARR7
ARR8
ARR9
CKI1

showed a characteristic poor growth phenotype with a small

yield of seeds (Fig. 8A). These growth phenotypes were very
His-kinases Type-A ARRs similar to those observed for plants carrying a dominant-
C t-Zeatin DMSO negative wooden leg (wol) allele of AHK4/CRE1 (Mähönen
0 30 60 90 30 60 90 (min)
ARR15 et al. 2000, Mähönen et al. 2006b). More importantly, the
Col
arr10/12 protoxylem specification in question was extremely accel-
ARR16 erated in the roots of arr1/10/12 even when the seedlings
Col were not pre-treated with BA, as previously demonstrated
arr10/12
for wol (Fig. 8B, C). These findings suggest that the
ARR6
Col cytokinin responses in plants are attenuated in situ in the
arr10/12 arr1/10/12 triple mutant as severely as in the wol mutant,
ARR7 as discussed further below.
Col
In the previous study (Mähönen et al. 2006a), in
arr10/12
AtHK1
addition to the wol mutant, another characteristic mutant
Col was employed to clarify their model (see Fig. 8B). This is a
arr10/12 null mutant allele (referred to as ahp6-1) of the AHP6 gene
ACT8
Col
arr10/12
set of histidine–aspartate phosphorelay-associated genes. Values
of mean FC in response to cytokinin were calculated for Col
(open rectangles) and arr10/12 (shaded rectangles). The genes
Fig. 6 Root-specific microarray analyses with the arr10/12 double for histidine kinases and type-A ARRs were analyzed, as indicated.
mutant. (A) Profiling of expression levels of 36 genes, which were (C) Expression of certain histidine–aspartate phosphorelay-
significantly up-regulated in Col by cytokinin (see Table 1, associated genes in response to cytokinin. RNA samples were
FC 42.5). For each gene, values of average expression levels prepared from roots of the Col and arr10/12 seedlings (14-day-old),
(triplicate sample) in the four kinds of RNA samples from Col after being treated with 20 mM BA for 30, 60 and 90 min.
and arr10/12 plants treated or not with cytokinin were normalized These samples were analyzed by semi-quantitative RT–PCR
with the GeneSpring software. The expression levels are indicated for transcripts of ARR15, ARR16, ARR6, ARR7, AtHK1 and ACT8,
by the intensities of colors (green, yellow and red), as specified as indicated. Several (at least three) different cycle conditions
at the bottom. (B) Microarray data for the expression levels of a were used, and those presented are quantitative representatives.
92 Cytokinin responses in Arabidopsis thaliana

A Cytokinin Col wol arr1/10/12

Procambium A
ARR10/12
AHKs
Protoxylem

B Col arr10/12 ahk3/4

B Cytokinin
Procambium
0 0.1 10 0 0.1 10 0 0.1 10

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AHKs ARR1/10/12
BA (mM)
(wol)
Protoxylem
AHP6
C BA (mM)
0 0.1 10
C Col ahp6

** ** **
Col
arr10/12

** ** **

wol arr1/10/12
ahk3/4

** ** **

Protoxylem Metaxylem
*
Fig. 7 Regulation of cell fate by cytokinin during vascular
development in the roots of arr10/12. (A) A hypothetical view of
the roles of ARR10 and ARR12 in regulation of cell fate by
cytokinin during vascular development in roots. This view is
principally based on the model proposed recently by Mähönen
et al. (2006). In the model, it was proposed that the differentiation
of protoxylem from procambium in roots is negatively controlled
by the AHK-mediated phosphorelay signal transduction in response
to cytokinin. Here, we speculated that ARR10 and ARR12 might be
implicated in this regulation of vascular development, as shown.
(B) Photographic presentation of seedlings (4-day-old) of Col,
arr10/12 and ahk3/4. They were grown on MS agar plates
containing the indicated concentrations of BA. Primary roots of
these seedlings were examined with special reference to the
differentiation of protoxylem from procambium. Yellow scale bars
indicate 5 mm, and red arrows indicate the approximate positions
where vascular tissues were examined in the following experi-
ments. (C) Confocal images of the differentiation of protoxylem
from procambium in the roots of Col, arr10/12 and ahk3/4.
Seedlings (see B) were stained with fuchsin to visualize both
protoxylem and metaxylem, as specified by yellow arrowheads and
asterisks. Note that the regions of roots examined are indicated in
(B) by red arrows. Yellow scale bars indicate 10 mm.
encoding a pseudo-type of AHPs. In this model, AHP6 was
demonstrated to be a negative regulator of the AHK-
dependent signaling pathway. Here the roots of these two
mutants (ahp6 and wol) were also examined with regard to
Protoxylem Metaxylem
Fig. 8 Regulation of cell fate by cytokinin during vascular
development in the roots of arr1/10/12. (A) Morphological views
of young plants (20-day-old) grown on MS agar plates. The pictures
are for Col, wol and arr1/10/12, as indicated. (B) A hypothetical
view of the roles of type-B ARRs in regulation of cell fate by
cytokinin during vascular development in roots. This view is
principally based on the model proposed recently by Mähönen
et al. (2006). In the model, it was proposed that the differentiation
of protoxylem from procambium in roots is negatively controlled
by the AHK-mediated phosphorelay signal transduction in response
to cytokinin. A homolog of phosphorelay intermediates
(named AHP6) was also implicated as a negative regulator. Here,
we speculated that ARR1/ARR10/ARR12 might be implicated as
positive regulators in this regulation of vascular development,
as shown. (C) Confocal images of the differentiation of protoxylem
from procambium in the roots of Col, ahp6, wol and arr1/10/12.
Other details are given in the legend to Fig. 7C.
root vascular development, in order to verify the experi-
mental conditions of this study (Fig. 8C). Consistent with
the model, the protoxylem specification was significantly
attenuated in the roots of ahp6, as compared with the case
of Col (note that no or only one protoxylem was seen in the
 Cytokinin responses in Arabidopsis thaliana
Cytokinin
IPT5 / 7 ARR15 / 16 PC
AtHK1
AHK2/3/4 ARR10/12
Root Differentiation PX
Root Elongation
In Vitro Shoot Initiation
Fig. 9 Summarized views of the roles of ARR10 and ARR12 in
the histidine–aspartate phosphorelay signal transduction in roots.
The findings of this study were collectively incorporated into the
schematic views. It should be noted that the view is intended to
be tentative in the sense that verification must await further
examinations. The details are discussed in the text.
roots of ahp6). On the other hand, the protoxylem
specification was extremely accelerated in the roots of
wol (note that many extra protoxylem files were seen),
as mentioned above. This finding for wol was explained by
the wol allele exerting a strong dominant negative effect on
its homologous AHK2 and AHK3 cytokinin receptors,
and, therefore, the cytokinin responses are severely
attenuated in the wol mutant plants because they virtually
completely lack the cytokinin receptor activities, with
phosphatase activity for AHPs remaining (Mähönen et al.
2006b). As a result, the growth of wol on MS plates is
severely retarded, with a poorly developed root system, as
seen in Fig. 8C. Taking these critical reference data
together, we showed for the first time that the cytokinin
responses in plants are attenuated in situ in the arr1/10/12
triple mutant as severely as in the wol mutant, and both
display marked phenotypes very similar to each other.
Implications
According to the well-documented scenario with
regard to cytokinin-mediated signal transduction
(Sheen 2002), certain members of the type-B ARR family
act as cytokinin-responsive transcription factors (Fig. 1).
This conceptual view was already supported by a number of
pieces of evidence from studies on the type-B ARR family
members through forward and reverse genetics (Imamura
et al. 2003, Tajima et al. 2004, Mason et al. 2005).
Nevertheless, further studies on these genes are currently
hampered by the fact that they apparently play very
redundant roles in plants (Imamura et al. 1998, Imamura
et al. 1999, Mason et al. 2004). As an attempt to overcome
this problem, here we focused on a pair of highly
homologous ARR10 and ARR12 genes (Fig. 2), and
established a new arr10 arr12 double mutant (Fig. 3).
Through such extensive forward genetics, the functions of
ARR10 and ARR12 were clarified with special reference to
the histidine–aspartate phosphorelay-mediated cytokinin
93
signaling in roots. The essence of these findings is
summarized in Fig. 9. In short, we demonstrated for the
first time that ARR10 and ARR12 are involved in the
AHK-dependent signaling pathway that modulates the
differentiation of root vascular tissues (i.e. protoxylem
specification). Furthermore, a newly constructed arr1/10/12
triple mutant showed striking phenotypes, which were very
similar to those displayed by wol and/or ahk2/3/4. Taken
together, we would like to propose that ARR10 and
ARR12, together with ARR1, play prominent (or pivotal)
roles in the AHK-dependent phosphorelay signaling in
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response to cytokinin in roots. As seen in Fig. 1, the views
presented in this study will also provide us with an insight
into cytokinin signaling in the model crop rice.
Materials and Methods
Plant materials and growth conditions
All the A. thaliana lines were in a Col background. Nucleotide
positions are reported relative to the inferred initiation codon (þ1).
To generate the ARR10 promoter::GUS reporter construct,
the genomic region between the 1,469 nucleotide position and
the the þ27 position of ARR10 was amplified using the primers
50 -agaaaaggagaggcacgg-30 and 50 -gacttcaatttcttgctcc-30 , and cloned
in-frame upstream of the b-glucuronidase (GUS) gene in the
binary vector pABH-HM2 [a derivative of pBI101 (Jefferson et al.
1987)]. To generate the ARR12 promoter::GUS reporter construct,
the genomic region between the 1,660 nucleotide position
and the þ3 position of ARR12 was amplified using the primers
50 -tactggatatcagatagatgtgg-30 and 50 -catcgtttttcaacagaccc-30 , and
cloned in-frame upstream of the GUS gene in the binary
vector pABH-HM2. These constructs were transformed into
A. tumefaciens strain C58C1, and then wild-type Arabidopsis
plants (Col) were transformed by vacuum infiltration procedures
(Bechtold 1993). arr10-5 is the SALK_098604 T-DNA insertion
line, whose mutation was determined to be located in the fifth exon
of the ARR10 coding sequence (at position þ1,513/þ1,527).
arr12-1 is the SALK_054752 T-DNA insertion line, whose
mutation was determined to be located in the third exon of the
ARR12 coding sequence (at position þ717/þ726). arr1-4 is
the SALK_001450 T-DNA insertion line, whose mutation was
determined to be located in the third exon of the ARR1 coding
sequence (at position þ1,033/þ1,034). arr10-5 arr12-1 double and
arr1-4 arr10-5 arr12-1 triple mutants were generated by crossing.
ahk3-3 cre1-12 is a gift from Dr. Tatsuo Kakimoto (Osaka
University). The wol allele of AHK4/CRE1 [AHK4/CRE1(T278I)]
is a gift from Dr. Yka Helariutta (University of Helsinki, Finland).
ahp6-3 is the SALK_058085 T-DNA insertion line, whose
mutation was determined to be located in the second exon of the
AHP6 coding sequence (at position þ436/þ437). Plants were
grown with 16 h light/8 h dark fluorescent illumination
at 228C on agar plates containing MS salts, 0.05% MES and
1% sucrose, pH 5.7 (described as MS agar plates for brevity),
unless otherwise noted. When RNA was prepared or the root
growth inhibition assay was carried out, plants were grown
under constant light conditions. If necessary, BA was added to
the agar plates.
94 Cytokinin responses in Arabidopsis thaliana
Expression analyses with GUS activities
Detection of GUS activity was carried out according to
the method described previously (Donnelly et al. 1999) with some
modifications. Five-day-old seedlings were first placed in 90%
acetone on ice for 15 min and in staining reaction buffer
solution [750 mg ml1 5-bromo-4-chloro-3-indolyl-b-d-glucur-
onide, 100 mM sodium phosphate (pH 7), 3 mM K3F3(CN)6,
10 mM EDTA, 0.1% NP-40] under moderate vacuum pressure
(500 mmHg) for 16 h at room temperature. They were then
transferred to 70% ethanol. After 30 min, they were placed in
destaining solution (6 vols. of ethanol, 1 vol. of acetic acid) for
41 h. The seedlings were then transferred to 70% ethanol again.
After 30 min, they were placed in chloral hydrate solution
(trichloroacetaldehyde monohydrate : glycerol : water ¼ 8 g : 1 ml :
2 ml) for 1 h. Samples were observed using a stereomicroscope
(MS420, Leica) with a cooled CCD camera (DC300F) or a
Nomarski microscope (E600, Nikon) with a digital camera
(COOLPIX990, Nikon).
Root growth inhibition assay
Seedlings grown for 8 d on vertically oriented MS agar plates
including appropriate concentrations of BA with 0.01% dimethyl-
sulfoxide (DMSO) were removed from the plates, and root lengths
were measured. Plants that had not germinated within 2 d of
culture were excluded from the analysis. An average length of roots
with standard deviation was calculated for each sample.
DNA microarray analysis
In order to analyze the primary genome-wide response to
cytokinin in roots, the wild type and the arr10/12 mutant grown on
vertically oriented MS agar plates for 2 weeks were treated with
20 mM t-zeatin or 0.02% DMSO (solvent for t-zeatin solution) for
1 h. These treated plant samples were divided into three portions,
from which RNA samples were prepared separately from roots of
seedlings with use of an RNeasy Plant Mini Kit (Qiagen, Valencia,
CA, USA). The quality of RNAs prepared was analyzed using a
Bioanalyzer 2100 (Agilent Technologies). Microarray analysis was
performed using a GeneChipÕ ATH1 Arabidopsis genome array
kit (Affymetrix, Santa Clara, CA, USA). Preparation of labeled
target cRNA, and subsequent purification and fragmentation were
carried out using one-cycle target labeling and control reagents
(Affymetrix). Double-stranded cDNA was prepared from 10 mg of
total RNA. Hybridization, washing, staining and scanning were
performed as described in the supplier’s protocol. A 10 mg aliquot
of fragmented cRNA was subjected to hybridization. It should be
noted that all the above manipulations were carried out
independently for each RNA sample. Data analysis was performed
using GeneChipÕ Operating Software (GCOS; Affymetrix) and
GeneSpring 7.3 (Agilent Technologies, Palo Alto, CA, USA).
Normalization per chip and per gene of the log2 values was
performed as recommended by the GeneSpring manual for
Affymetrix gene chips. In order to clarify cytokinin-induced
genes, first, genes assigned to P (present) in any cytokinin-treated
sample of the wild type (triplicate) were selected according to the
Affymetrix flag procedure. Secondly, genes in which values of the
standard deviation of the normalized expression level in cytokinin-
treated samples of wild type exceeded 25% of the mean were
excluded from the analysis. Then the values of the mean normal-
ized expression level of cytokinin-treated samples were compared
with those of DMSO-treated samples in the wild type according to
the FC procedure, and genes whose expression in the wild type was
increased (mean FC 42.5) in response to cytokinin were selected.
In order to clarify cytokinin-repressed genes, first, genes assigned
to P (present) in any DMSO-treated sample of the wild type
(triplicate) were selected according to the Affymetrix flag
procedure. Secondly, genes in which values of the standard
deviation of the normalized expression level in cytokinin-treated
samples of wild type exceeded 25% of the mean were excluded
from the analysis. Then the values of the mean normalized
expression level of cytokinin-treated samples were compared with
those of DMSO-treated samples in the wild type according to the
FC procedure, and genes whose expression in the wild type was
decreased (mean FC 50.4) in response to cytokinin were selected.
RT–PCR analysis
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The RNA PCR Kit (AMV) Version 2.1 (TAKARA BIO
INC.) was used according to the instructions. Transcripts from
arr10-5 and arr12-1 were analysed by RT–PCR with saturating
numbers of PCR cycles on RNA prepared from the wild type
and plants carrying each single mutation by using the gene-specific
primers indicated in Fig. 3A (primer a, 50 -GACGGCGATGACTA
TGGAGC-30 ; primer b, 50 -GTGAGTCAATAGCCGCCCTG-30 ;
primer c, 50 -GGAGTTTCTCGTATGCATGTTCCG-30 ; primer d,
50 -CATCCCGGGATGCGTGGGAGTAATAATAATGGTGAT
AAGAG-30 ; primer e, 50 -GACCCCGGGGTCCTAAATTGCTA
GAACCAGTGGG-30 ). In the case of semi-quantitative RT–PCR,
it should be noted that the PCR cycles were varied from one RNA
sample to another, and several different cycles (at least three) were
adopted for a given RNA sample (from 20 cycles up to 32 cycles),
in order to amplify double-stranded DNA in a semi-quantitative
manner. In such a semi-quantitative RT–PCR, the primers
used were: ARR6 (50 -CAAAGTTTGGATCACCGGATCC-30
and 50 -CTGCGAGTGAACAGGGTAGAC-30 ), ARR7 (50 -CGG
TTGGTGAGGTCATGAGG-30 and 50 -GGTTTTGCTAAGGT
CTTGGCCTC-30 ), ARR15 (50 -CATCTGTTTTGTTGTTTACT
TTCCCGAGAG-30 and 50 -GGTGAGCATTAGAATCTAGAC
TTACATAGTTG-30 ), ARR16 (50 -CAGTTACTTGACATTTAA
GGAACAAAGGAG-30 and 50 -CACAATGAAAACGTTTCA
TGGTTAGGGC-30 ) and AtHK1 (50 -CTTCGTGGATGGTGCG
AGAAC-30 and 50 -CGCCTTTGTTGCCTCATATCCATCC-30 ).
Confocal microscopic analyses of xylem differentiation
Fluorescent staining of root vasculature tissues was carried
out as described previously (Mähönen et al. 2000). In brief, young
seedlings (4-day-old) were cleared by incubation with acidified
methanol [10 ml of methanol, 2 ml of concentrated HCl (37%),
38 ml of H2O] at 568C for 15 min. They were transferred into a
basic solution (7% NaOH in 60% ethanol), and incubated for
15 min at room temperature. They were then rehydrated by
successive treatment with decreasing concentrations of ethanol
(first 40% ethanol, then 20%, and finally 10%). They were stained
by 0.01% basic fuchsin solution for 3 min, followed by de-staining
with 70% ethanol. The samples were then rehydrated, as described
above. An equal amount of 50% glycerol was added to the
sample solutions, and they were incubated for 30 min. They
were examined with a laser scanning confocal microscope
(FV500, Olympus, 488 nm emission with an argon ion laser, and
568 nm detection).
Acknowledgments
We thank Dr. Tatsuo Kakimoto (Osaka University) for
providing the ahk3-3 cre1-12 double mutant seeds. We also thank
Dr. Yka Helariutta (University of Helsinki, Finland) for providing
wol mutant seeds. Thanks are also due to Dr. Yutaka Sato for kind
 Cytokinin responses in Arabidopsis thaliana
support relating to histological techniques. This study was
supported by Grants-in-Aid for scientific research on a priority
area, ‘Molecular Basis of Axis and Signals in Plant Development’
(Grant No.17027014 to T.Y.) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
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(Received October 24, 2006; Accepted November 17, 2006)