Báo cáo khoa học: The purine derivative PI-55 blocks cytokinin action via receptor inhibition docx

The purine derivative PI-55 blocks cytokinin action via
receptor inhibition
Luka
´
s
ˇ
Spı
´chal
1
, Toma
´
s
ˇ
Werner
2
, Igor Popa
1
, Michael Riefler
2
, Thomas Schmu
¨
lling
2
and Miroslav Strnad
1
1 Laboratory of Growth Regulators, Institute of Experimental Botany, AS CR & Palacky
´
University, Olomouc, Czech Republic
2 Institute of Biology ⁄ Applied Genetics, Free University of Berlin, Germany
Cytokinins, which comprise an important class of
plant hormones, are chemically classified as N

6
-substi-
tuted derivatives of adenine with either isoprenoid (e.g.
zeatin) or aromatic [e.g. 6-benzylaminopurine (BA)]
sidechains [1] (Fig. 1). Cytokinins regulate diverse
processes during growth and development. The histi-
dine kinases cytokinin response 1 (CRE1) ⁄ Arabidopsis
histidine kinase (AHK)4, AHK3 and AHK2 have been
identified as cytokinin receptors in Arabidopsis [2,3],
which feed the signal into a two-component signaling
pathway. Cytokinin receptors have also been identified
from other species, including maize [4], the legumes
Medicago truncatula [5] and Lotus japonicus [6,7], and
rice [8]. Genetic analyses of Arabidopsis cytokinin
receptor mutants has assigned functions to the recep-
tors in the regulation of shoot growth, leaf senescence,
seed size and germination, and root elongation and
branching. In addition, analysis of loss-of-function and
gain-of-function mutations in legume cytokinin recep-
tors has revealed their direct involvement in nodule
organogenesis [5–7].
Chemical inhibitors of cytokinin action would be
very potent tools to study the mechanism of cytokinin
perception and signal transduction. Furthermore, they
Keywords
anticytokinin; cytokinin; cytokinin receptor;
cytokinin receptor antagonist; hormone
signaling
Correspondence
L. Spı

´
chal, S
ˇ
lechtitelu
˚
11, CZ-78371
Olomouc, Czech Republic
Fax: +420 58 5634870
Tel: +420 58 5634855
E-mail:
Website:
(Received 26 August 2008, revised 24
October 2008, accepted 3 November 2008)
doi:10.1111/j.1742-4658.2008.06777.x
One of several potential approaches to study mechanisms of action of bio-
logically active compounds is to develop their agonists and antagonists. In
the present study, we report the identification of the first known molecule
antagonizing the activity of the plant hormone cytokinin at the receptor
level. This compound, 6-(2-hydroxy-3-methylbenzylamino)purine, desig-
nated PI-55 in the present study, is structurally closely related to cytokinin
6-benzylaminopurine, but substitutions at specific positions of the aromatic
side chain strongly diminished its cytokinin activity and conferred antago-
nistic properties. PI-55 competitively inhibited the binding of the natural
ligand trans-zeatin to the Arabidopsis cytokinin receptors cytokinin
response 1 (CRE1) ⁄ Arabidopsis histidine kinase (AHK) 4 and AHK3 and
repressed induction of the cytokinin response gene ARR5:GUS. Genetic
analysis revealed that CRE1 ⁄ AHK4 is the primary target of PI-55. Cyto-
kinin bioassays also demonstrated the anticytokinin effect of PI-55 in several
other species. Furthermore, we show that PI-55 accelerated the germination
of Arabidopsis seeds and promoted the root growth and formation of lateral

roots, thus phenocopying the known consequences of a lowered cytokinin
status and demonstrating its potential to inhibit cytokinin perception
in planta. PI-55 is the first example for the targeted development of a cyto-
kinin antagonist and represents an initial step for the preparation of cyto-
kinin antagonists with broad activity and reduced agonistic properties.
Abbreviations
AHK, Arabidopsis histidine kinase; BA, 6-benzylaminopurine; CDK, cyclin dependent kinase; CRE1, cytokinin response 1; DAG, days after
germination; PI-55, 6-(2-hydroxy-3-methylbenzylamino)purine.
244 FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS
would be expected to influence plant growth and devel-
opment and might thus possess a potential for agricul-
tural application. Subsequent to the discovery of
cytokinins, several attempts have been made to identify
compounds with anticytokinin properties. Diverse
adenylate and non-adenylate compounds with agonis-
tic and antagonistic effects have been synthesized.
Some of them were classified as anticytokinins because
of their strong inhibitory effect on cytokinin-induced
responses in various bioassays. N
6
-sidechain cytokinin
analogues with alterations in the purine ring were pre-
pared in the 1970s, based on the rationale that cyto-
kinin antagonists are likely to have structural similarities
to cytokinins because such features should allow them
to compete for the same receptor(s) but render them
ineffective as cytokinins [9]. For example, heterocyclic
compounds sharing cytokinin structural motifs, such
as 7-substituted 3-methyl-pyrazolo[4,3-d]pyrimidines
[10,11], 4-substituted pyrrolo[2,3-d]pyrimidines and

their 7-glucosides [12,13], and 7-deaza analogues of
2-methylthioadenine cytokinins [14], were prepared
and their activities were tested in a callus growth test.
Some of the compounds had strong growth-retarding
effects and, because the addition of cytokinins reversed
the growth arrest, these results were interpreted as
evidence indicating that these compounds were specific
anticytokinins sharing the same site of action as active
cytokinins [9].
Following observations that some phenylureas struc-
turally unrelated to cytokinins can exhibit cytokinin-
like activity, cytokinin antagonists were sought among
urea derivatives. Several compounds were identified
based on the reduction of cytokinin-induced effects,
such as chlorophyll retention, radish leaf expansion
and callus growth [15]. Other anticytokinins with non-
adenine structures were sought based on considerations
of the biosterism between N
6
-substituted adenine,
phenylurea cytokinins and the herbicidal inhibitors of
photosystem II, s-triazines and N-arylcarbamates [16].
It was concluded that phenylureas, s-triazines and
N-arylcarbamates share the same site of action: a cyto-
kinin receptor localized in the chloroplast [9]. How-
ever, the effects of cytokinin antagonists have been
defined primarily in tobacco callus assays, and hence
their antagonistic properties in other plant systems are
uncertain [9]. Moreover, direct proof that the molecu-
lar target of these compounds is a cytokinin receptor

has been hindered by a lack of knowledge of cytokinin
receptors and signaling. However, the recent identifica-
tion of Arabidopsis cytokinin receptors [2,3] has
allowed direct examination of the true mode of action
of known anticytokinins. We have recently shown that
representatives of the anticytokinins described above
are not competitive inhibitors of cytokinin receptors
but inhibit cell cycle progression (i.e. the activity used
for their classification as anticytokinins) through a dif-
ferent mechanism. Indeed, some compounds were
shown to inhibit cyclin dependent kinases (CDKs) and
also cause a number of cellular changes typical of
known CDK inhibitors [17].
The identification of cytokinin receptors enabled us
to employ a novel approach. We followed the assump-
tion that a compound possessing true antagonistic
activity should compete with the natural ligands for
the receptor(s) and block cytokinin action in vivo, but
should not have intrinsic cytokinin activity in cyto-
kinin bioassays, and neither activate the cytokinin
signaling pathway, nor be cytotoxic or inhibit CDKs.
Based on these criteria, we screened a library of
synthetic compounds derived from cytokinins in a
receptor-based assay. We have identified a BA deriva-
tive, 6-(2-hydroxy-3-methylbenzylamino)purine, desig-
nated PI-55 in the present study (Fig. 1), and describe
its antagonistic effects in various cytokinin-mediated
processes in the model plant Arabidopsis thaliana and
other species.
Results

PI-55 reduces binding of the natural ligand to
cytokinin receptors
In the search for the cytokinin antagonists that genu-
inely act at the receptor level, we focused on structural
derivatives of cytokinins having substitutions in
specific positions that diminish cytokinin-like activity.
We employed transformed Escherichia coli strains
expressing the cytokinin receptors CRE1 ⁄ AHK4 and
AHK3 and the cytokinin-activated reporter gene
cps::lacZ [4,18,19], and used three classical cytokinin
Fig. 1. Structures of active cytokinins and the newly identified
anticytokinin PI-55. tZ, trans-zeatin.
L. Spı
´
chal et al. PI-55 blocks cytokinin action
FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS 245
bioassays (tobacco callus, wheat senescence and
Amaranthus bioassays) to select compounds from
among > 400 synthetic cytokinin derivatives that are
not sensed by the receptors and, in addition, have no
activity in the cytokinin bioassays. These compounds
were further tested in binding assays with isolated
E. coli membranes containing CRE1 ⁄ AHK4 or AHK3
[17] for their ability to compete for receptor binding
with the radiolabeled natural ligand trans-zeatin,
employing unlabeled trans-zeatin and adenine as posi-
tive and negative controls, respectively. An example of
the strategy for the selection of the candidate cytokinin
antagonist is summarized in Table 1. It is well known
that a hydroxyl group placed in the ortho- position, in

contrast to the meta- position, of the aromatic side
chain of the highly active cytokinin BA strongly
decreases its receptor sensing and biological activity in
cytokinin bioassays (Table 1) [17,20]. Interestingly, the
situation is reversed when a methyl group is substi-
tuted in these positions (Table 1) [21]. Simultaneous
substitution by both hydroxyl and methyl groups (in
the ortho- and meta-positions, respectively) minimized
the ability of the compound to activate the cytokinin
signaling pathway and reduced its cytokinin-like activ-
ity in bioassays (Table 1). The disubstituted BA, desig-
nated PI-55 in the present study (Fig. 1), did not
activate the CRE1 ⁄ AHK4-mediated signaling pathway,
even at a concentration 500-fold greater (50 lm) than
that required for receptor activation by trans-zeatin
(Fig. 2A). Similarly, up to 10 lm, PI-55 no activation
of AHK3 was observed, although the highest concen-
tration induced a weak response slightly greater than
that induced by the negative control substance adenine
(Fig. 2B). AHK3 has been shown previously to have
broad ligand specificity [19], which might explain this
response. Importantly, in our in vitro system, PI-55
reduced the binding of [2-
3
H]zeatin to both receptors
in a dose-dependent manner, with IC
50
values that
were only four-fold higher than those of the cytokinin
BA, whereas adenine was unable to compete even at

1000-fold excess (Fig. 2C; see also Fig. S1A). Thus, a
slight modification of the cytokinin structural motif led
to a compound that does not activate the cytokinin
signaling pathway, but competes for receptor binding
with only a fourfold lower efficacy than widely used
cytokinin BA and approximately 200-fold lower than
the strongest natural cytokinin trans-zeatin. Analysis
of double-reciprocal plots showed that PI-55 was com-
petitive towards trans-zeatin with K
i
values of
1.7 ± 0.9 lm and 10.6 ± 0.7 lm in the CRE1 ⁄ AHK4
(Fig. 2D) and AHK3 assays (see Fig. S1B), respec-
tively.
PI-55 inhibits the activation of a cytokinin
primary response gene
To support our finding that PI-55 truly competes with
cytokinin for binding to cytokinin receptors, we next
determined whether PI-55 blocks cytokinin signal
transduction in planta. We recorded the response of
the ARR5:GUS reporter gene, which is known to be
rapidly upregulated by cytokinin [22,23]. PI-55
decreased the blue staining caused by ARR5:GUS
expression following treatment by 2.5 lm BA in the
root and shoot (Fig. 3A). The quantitative evaluation
of the effect of PI-55 on BA-induced ARR5:GUS
expression demonstrated its competitive inhibitory
effect (Fig. 3B). The competitive mechanism of this
inhibition was judged from the previous findings that
PI-55 directly competes with BA for binding to the

cytokinin receptors (Fig. 2C; see also Fig. S1A). PI-55
alone only weakly induced ARR5:GUS, probably due
to weak activation of AHK3 (Fig. 2B) and eventually
AHK2. To investigate in more detail to what extent
Table 1. Development of a cytokinin receptor antagonist. The ability of different substituted BAs to activate the cytokinin signaling pathway
through the receptors AHK3 and AHK4 in a bacterial assay and to block the binding of radiolabeled natural ligand to the receptors, as well as
the biological activity in classical cytokinin bioassays, are compared. Adenine is shown as the negative control. The details of the assays are
described in the Experimental procedures. For the semi-quantitative evaluation: +++, high; ++, moderate; +, low; –, none.
Compound
Substituent
Activation of signal-
ing pathway
Competition for
receptor binding
Activity in
cytokinin
bioassays
R1 R2 AHK3 AHK4
BA H H +++ +++ +++ +++
N
H
N
N
N
NH
R2
R1
2-OH-BA OH H + ) ++
3-OH-BA H OH +++ +++ +++ +++
2-Me-BA CH

3
H ++ + + +++
3-Me-BA H CH
3
+ ) +++
2-OH-3-Me-BA (PI-55) OH CH
3
))+++ )
Ade ))) )
PI-55 blocks cytokinin action L. Spı
´
chal et al.
246 FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS
different AHK proteins are sensitive to inhibition by
PI-55, we introduced the ARR5:GUS gene construct in
the background of all three AHK double loss-of-func-
tion mutants [24] and examined them in competitive
assays. In this experiment, the seedlings were grown in
the presence of 1 lm BA and 1 or 5 lm PI-55. As
shown in Fig. 3C, the strongest reduction of ARR5:
GUS activity was observed with 5 lm PI-55 in the
ahk2 ahk3 mutant, in which the b-glucuronidase
activity was significantly reduced by 40% (calculated
from the values of b-glucuronidase specific activ-
ity shown in Fig. 3C) compared to statistically not
Fig. 2. Effects of PI-55 on the activity of
cytokinin receptors and cytokinin binding.
(A, B) Comparison of the sensitivity of
CRE1 ⁄ AHK4 (A) and AHK3 (B) to the cytoki-
nins trans-zeatin (tZ), BA, PI-55 and adenine

(Ade) in a bacterial reporter assay. The
compounds were tested at the indicated
concentrations; 0.5% dimethylsulfoxide
(DMSO) was used as control solvent. Error
bars show the SD (n = 3). (C) Representa-
tive example of competitive binding assay
with CRE1 ⁄ AHK4 containing E. coli mem-
branes. Binding of 2 n
M [2-
3
H]tZ was
assayed in the presence of various concen-
trations of PI-55, unlabelled BA and tZ (posi-
tive control) and adenine (Ade, negative
control). (D) Representative example of
Lineweaver–Burk plot of 2 n
M [2-
3
H]tZ bind-
ing to CRE1 ⁄ AHK4-containing E. coli
membranes in the presence of different
concentrations of PI-55. The inset shows a
secondary plot of slopes versus inhibitor
concentration.
A
C
B
Fig. 3. Effect of PI-55 on expression of a
cytokinin response gene. (A) Staining for
b-glucuronidase activity in shoots (upper

row) and roots (lower row) of 9-day-old
ARR5:GUS-expressing transgenic Arabidop-
sis seedlings treated with 2.5 l
M BA in the
presence or absence of 5 l
M PI-55. (B)
Quantitative fluorometric measurement of
PI-55 dose-dependent suppression of cyto-
kinin-induced expression of ARR5:GUS in
3-day-old seedlings. (C) Quantitative evalua-
tion of b-glucuronidase activity 3 DAG in ahk
double receptor mutants harboring ARR5:-
GUS after incubation with BA and PI-55.
Dimethylsulfoxide (0.1%) was used as con-
trol solvent. Error bars show the SD (n = 2).
Asterisks indicate a statistically significant
decrease of induction of cytokinin-induced
ARR5:GUS expression (Student’s t-test,
P < 0.05).
L. Spı
´
chal et al. PI-55 blocks cytokinin action
FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS 247
significant reduction in the ahk2 ahk4 and ahk3 ahk4
seedlings. The results confirmed that PI-55 most
strongly competes for cytokinin binding at the
CRE1 ⁄ AHK4 receptor, and that its impact on the sig-
nal transduction mediated through AHK2 and AHK3
is probably weaker. 1 lm PI-55 alone induced 26%,
38% and 66% of the BA-induced reporter gene

activity in the ahk2 ahk3, ahk2 ahk4 and ahk3 ahk4
mutant plants, respectively, which is consistent with
the result of the bacterial assay that PI-55 may act also
as a weak agonist, in particular with AHK3, and ⁄ or
may point to another recognition systems for cyto-
kinins leading to expression of cytokinin responsive
genes.
PI-55 affects root growth and stimulates root
branching
Cytokinins are known to be negative regulators of root
growth [24–27]. To study whether PI-55 is able to
repress this cytokinin effect, we grew wild-type Arabid-
opsis seedlings on media containing BA and various
concentrations of PI-55. PI-55 alone did not affect the
elongation of the primary root of wild-type Arabidop-
sis seedlings; positive and, importantly, negative effects
were not observed even at 1 lm. By contrast, 1 lm BA
almost completely inhibited primary root growth (see
Fig. S2). However, when 1 lm PI-55 was applied to
the growth media of ahk2 ahk3 double mutants, a
significant increase (approximately 15%) in the length
of the primary roots was observed compared to the
untreated control (Fig. 4A). No significant changes in
elongation of the primary root were found with other
two receptor double mutants (data not shown). This
result is consistent with a predominant activity of
PI-55 on CRE1 ⁄ AHK4 and indicates that this receptor
has a regulatory function in the control of primary
root elongation.
A clear antagonistic effect of PI-55 on root branch-

ing was observed 14 days after germination (DAG).
Even at 1 nm, BA caused a strong inhibition of lateral
root formation, and its effect was more pronounced at
higher concentrations. An equivalent concentration of
PI-55 (1 nm) almost completely reversed the suppres-
sion of lateral root formation by BA (Fig. 4B). Inter-
estingly, PI-55 had a positive effect on root branching
when applied alone (Fig. 4B), indicating that it also
suppressed the activity of endogenous cytokinins. Fur-
ther investigations with receptor double mutant plants
showed that, although 10 nm PI-55 had only a slightly
positive effect on formation of lateral roots in ahk3
ahk4 seedlings, and no effect in ahk2 ahk4 seedlings, it
caused ahk2 ahk3 seedlings to form a significantly
higher number of lateral roots than untreated controls
(Fig. 4C). This result again points to activity of PI-55
on CRE1 ⁄ AHK4 and indicates its regulatory function
in the control of lateral root formation and root
response to exogenous cytokinin.
Fig. 4. Effect of PI-55 on primary root length and lateral root formation in Arabidopsis seedlings. (A) Increase in the length of the primary
roots observed in ahk2 ahk3 double mutant seedlings (6 DAG) grown on medium containing 1 l
M PI-55. (B) The number of lateral roots
formed by wild-type Arabidopsis seedlings (11 DAG) in the presence or absence of BA and ⁄ or PI-55. (C) The increase in the number of
lateral roots observed in wild-type and ahk double mutant seedlings (11 DAG) grown on medium containing 10 n
M PI-55. Error bars show
the SD (n = 20). Asterisks indicate statistically significant differences from the untreated controls (Student’s t-test, P < 0.05).
PI-55 blocks cytokinin action L. Spı
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248 FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS

PI-55 accelerates seed germination
Recently, it was demonstrated that cytokinin-deficient
Arabidopsis seeds or seeds with reduced cytokinin per-
ception germinate faster than wild-type seeds [24]. In
the present study, we tested the effect of PI-55 on ger-
mination. After sowing and pretreatment in the dark
at 4 °C, wild-type Arabidopsis seeds were transferred
to the light and ambient temperature. After 30 h, more
than 60% of seeds sown on medium containing 1 nm
PI-55 were germinating, which was twice as many than
on control medium without PI-55. The proportion of
germinating seeds was further enhanced at higher PI-
55 concentrations, and 80% of seeds had germinated
when 10 nm PI-55 was present in the medium (Fig. 5).
PI-55 antagonizes cytokinin effects in bioassays
with different plant species
Several classical cytokinin bioassays based on biologi-
cal responses to exogenous cytokinins were used to
evaluate the anti-cytokinin potential of PI-55 in other
plant species. The callus bioassay is based on the ability
of cytokinins to induce cell division in presence of
auxin [28]. Cytokinin-dependent tobacco (Nicotiana
tabacum) callus was grown on medium supplemented
with various concentrations of PI-55 in the presence or
absence of 0.5 lm BA and the callus biomass was
scored after 30 days. 0.5 lm BA was found to be
optimal for maximal callus growth. PI-55 did not
significantly influence the growth of the calli when
applied alone. However, the growth stimulated by BA
was effectively inhibited by increasing concentrations of

PI-55, and concentrations as low as 10 lm caused total
inhibition of callus growth (Fig. 6A). It is possible that
such inhibitory effects on cell proliferation could be
due to the inhibition of proteins involved in cell
division, as previously shown for some pyrazolo[4,3-
d]pyrimidines and pyrrolo[2,3-d]pyrimidines, which
behave as anticytokinins in this bioassay [17], rather
than by competing with cytokinins for a common bind-
ing site on a receptor protein. However, PI-55 is not
cytotoxic and did not inhibit proliferation of human
cancer cell lines (G361, HOS and MCF7) and ⁄ or cyto-
kinin-dependent tobacco callus, even at concentrations
of 50 and 100 lm, respectively (see Table S1).
Another standard cytokinin bioassay is based on
cytokinin-stimulated delay of senescence in excised
leaves of wheat (Triticum aestivum), in which retention
of chlorophyll is measured after 4 days of incubation
in the dark [20]. As shown in Fig. 6B, 1 lm PI-55
reduced the retention of chlorophyll induced by 1 lm
BA, and its effect was more pronounced at 10 lm.
PI-55 had a limited effect when applied alone, but an
agonistic effect was observed at higher concentrations
(data not shown). A similar result was obtained in a
bioassay that is based on cytokinin-induced betacyanin
formation in hypocotyls of Amaranthus caudatus in the
dark [29]. Also in this assay, PI-55 showed an antago-
nistic effect at micromolar concentrations (Fig. 6C),
whereas higher concentrations of the compound had
weak cytokinin-like activity (Fig. 6C). These results
revealed that PI-55 has antagonistic activity to exo-

genously applied cytokinins in different bioassays in
various species.
Discussion
We identified a structural derivative of the aromatic
cytokinin BA, PI-55, as the first known anticytokinin
that acts at the receptor level. Specific substitutions in
ortho- and meta- positions of the aromatic sidechain of
BA resulted in altered perception by the cytokinin
receptors CRE1 ⁄ AHK4 and AHK3 in a bacterial
assay and isolated membranes of these bacteria.
Importantly, these substitutions did not affect the abil-
ity of the compound to compete for binding with the
natural ligand trans-zeatin (Fig. 2). Hence, only minor
changes in the aromatic cytokinin structure converted
the agonist BA into an antagonist. We propose that
PI-55 binds in a similar fashion to the receptor as does
BA, but does not induce the conformational change
Fig. 5. Effect of PI-55 on germination of Arabidopsis seeds. Wild-
type Arabidopsis seeds were incubated on MS medium containing
varying concentrations of PI-55. Error bars show the SD (n = 3).
DMSO, dimethylsulfoxide.
L. Spı
´
chal et al. PI-55 blocks cytokinin action
FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS 249
necessary to activate the receptor and initiate signaling,
as suggested previously by a hypothetical concept of
cytokinin action [9].
The antagonistic role of PI-55 during the early steps
of cytokinin signal transduction was confirmed

in planta in experiments showing that PI-55 inhibits
the cytokinin-induced activation of a cytokinin-respon-
sive marker gene. This contrasts with the inability of
previously described putative cytokinin antagonists,
which do not reduce the cytokinin-dependent activa-
tion of this reporter gene [17]. Consistently, it was
shown that these compounds do not interact with
cytokinin receptors of Arabidopsis but interfere at a
later stage with cytokinin action, namely by block-
ing the cell cycle through inhibition of CDK proteins
[17].
The results obtained in the present study further
support the hypothesis that the receptors have different
structural requirements for the stereochemical or phys-
icochemical features of the ligands. Whereas
CRE1 ⁄ AHK4 was not activated by PI-55 even at a
concentration 500-fold higher than that required for
receptor activation by trans-zeatin, AHK3 showed par-
tial activation at high concentrations of PI-55. This is
in accordance with results of a study of ligand specific-
ities of these two receptors [19] and of a screen for
cytokinin activities of various substituted BAs [21]. It
was shown that only a few tested BA derivatives were
able to activate CRE1 ⁄ AHK4 significantly, but most
of them were recognized by the AHK3 receptor, albeit
less efficiently than trans-zeatin [21]. The ligand speci-
ficity of AHK2 has not been described yet. However,
the results obtained based on PI-55 induction of cyto-
kinin reporter expression in ahk3 ahk4 mutant propose
that AHK2 also differs in ligand binding properties

from other two cytokinin receptors.
The activities of PI-55 in wild-type Arabidopsis
plants are in accordance with the results obtained in
several studies considering the consequences of geneti-
cally caused alterations of cytokinin signaling or
metabolism. Collectively, these studies, which involved
receptor loss-of-function mutations [19], mutations of
signaling proteins [30] and cytokinin-synthesizing genes
[31], or caused cytokinin-deficiency by enhanced cyto-
kinin degradation [25,32], showed that cytokinin acts
as a negative regulator of root growth. Consistently,
the presence of PI-55 in the growth medium led to
enhancement of the growth of root systems of
wild-type and receptor double mutant plants (Fig. 4).
Furthermore, PI-55 caused a more rapid germination
of Arabidopsis seeds (Fig. 5), which is also a character-
istic of seeds from plants with a reduced cytokinin sta-
tus [24]. The observation that PI-55 primarily blocks
the CRE1 ⁄ AHK4 receptor is in good agreement with
the finding that CRE1 ⁄ AHK4 is the receptor that
plays the main role in the control of germination by
cytokinin [24]. PI-55 interfered with cytokinin action in
various bioassays in other species, indicating that it
might be widely applicable and that a similar cytokinin
sensing mechanism to those mediated by CRE1 ⁄ AHK4
from Arabidopsis might be active in these species.
Taken together, the results obtained in the present
study indicate that PI-55 acts as an inhibitor of cyto-
kinin receptors and antagonizes cytokinin action
in vivo. Hence, in studies on the functions of cytokinins,

the compound can comprise a modulator of endo-
genous cytokinin activity as an alternative to genetic
approaches. In addition, it may have interesting
Fig. 6. Antagonistic effect of PI-55 in standard cytokinin bioassays. BA was used as the active cytokinin and its effect was studied in the
presence and absence of indicated concentrations of PI-55. (A) Effect of PI-55 on cytokinin-dependent growth of tobacco callus. (B) Effect of
PI-55 on cytokinin-induced retention of chlorophyll in excised wheat leaves. (C) Effect of PI-55 on cytokinin-stimulated betacyanin formation
in Amaranthus seedlings in the dark. Values represent the difference between absorption at 537 and 620 nm. Error bars show the SD
(n = 5).
PI-55 blocks cytokinin action L. Spı
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chal et al.
250 FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS
applications as a growth regulator for modifying traits
of crop plants. Synthetic compounds interfering with
the biosynthesis, metabolism or translocation of auxin,
gibberellin and ethylene are commonly used in basic
studies of the functions of these hormones. Some of
them are commercially used, such as inhibitors of eth-
ylene perception in controlling longevity in ornamental
crops [33]. Previously, such compounds have not been
available for cytokinin research. PI-55 is the first
example of the targeted development of a cytokinin
antagonist. Genetic and biochemical analyses revealed
that the primary target of PI-55 in Arabidopsis is
CRE1 ⁄ AHK4. The compound showed weak agonistic
activity resulting from the partial agonistic interference
with the other two cytokinin receptors. This corroborates
the differences in cytokinin binding sites of individual
receptors and indicates that the preparation of a
‘universal’ cytokinin receptor antagonist will not be an

easy task. PI-55 represents the first step demonstrating
the feasibility of preparing such compounds. However,
further steps (e.g. modulation of PI-55 structure) have to
be undertaken to broaden the antagonistic interference
and reduce the agonistic activity.
Experimental procedures
Chemicals
Synthesis of PI-55 is described in Doc. S1. The identity and
purity of the synthesized compound was confirmed by
elemental and melting point analyses, analytical TLC,
HPLC and MS and NMR analysis (for data, see Support-
ing information). trans-zeatin and BA were obtained from
Olchemim Ltd (Olomouc, Czech Republic). Radiolabeled
trans-zeatin ([2-
3
H]zeatin) was obtained from J. Hanus
ˇ
(Isotope Laboratory, Institute of Experimental Botany, AS
CR, Prague, Czech Republic).
Plant material and growth conditions
Arabidopsis plants harboring the ARR5:GUS reporter gene
construct and the Arabidopsis receptor double mutants have
been described previously [22,24]. For analysis of ARR5:-
GUS activity in the ahk mutant background, the ARR5:-
GUS marker gene was introgressed into the three different
ahk Arabidopsis double mutant lines. Arabidopsis thaliana
ecotype Columbia (Col-0) was used as a wild-type control.
For in vitro assays, seeds were surface-sterilized and sown
on half-strength Murashige & Skoog medium including
vitamins (Duchefa, Haarlem, The Netherlands) supple-

mented with 1% (w ⁄ v) sucrose, 0.05% (w ⁄ v) MES-KOH
(pH 5.7) and 1.1% agar. After cold pretreatment at 4 ° C
for 3 days in the dark, the seedlings were grown under
long-day conditions (16 : 8 h light ⁄ dark photoperiod) at
22 °C in a growth chamber unless otherwise stated. In the
classical cytokinin bioassays, cytokinin-dependent tobacco
(N. tabacum L. cv. Wisconsin 38) callus, seeds of winter
wheat (T. aestivum L. cv. Hereward) and Amaranthus
(A. caudatus L. var. atropurpurea) were used as described
below and as described previously [20].
Bacterial cytokinin assays
E. coli KMI001 strains harboring plasmids pIN-III-AHK4
and pSTV28-AHK3 [3,18] were used in the bacterial cytoki-
nin assays, which were performed as described previously
[19].
Fractionation of E. coli and microsome-binding
assays
CRE1 ⁄ AHK4- and AHK3-expressing E. coli strains [3,18]
were grown until D
600
of  1 was reached at 25 °C and
then fractionated into periplasmic, cytoplasmic and mem-
brane fractions. Fractionation and binding assays with
membranes were carried out as described previously [34].
Arabidopsis ARR5:GUS reporter gene assay
This assay was carried out as described previously [23], with
slight modifications. For quantitative assays, ARR5:GUS
seedlings were grown for 2–3 days (22 °C, 16 : 8 h light ⁄
dark photoperiod) in six-well plates (Techno Plastic Prod-
ucts, Trasadingen, Switzerland) and then cytokinin, PI-55

or control solvent (dimethylsulfoxide, final concentration
0.1%) were added to the desired final concentration. The
seedlings were then incubated for 17 h at 22 °C in the dark.
Histochemical b-glucuronidase analysis was performed with
9-day-old seedlings as described previously [25].
Root assay
Arabidopsis wild-type and receptor double-mutant seedlings
were grown on vertical plates in the presence of BA and ⁄ or
PI-55 or control solvent (0.1% dimethylsulfoxide). After
6 DAG, plates were photographed and the length of the
primary roots was scored using scion image software
(Scion Corp., Frederick, MD, USA). The number of lateral
roots was scored 11 DAG under a stereomicroscope.
Twenty plants for each genotype and treatment were
analyzed in the both cases.
Seed germination assay
This assay was performed under long-day conditions
(16 : 8 h light ⁄ dark photoperiod) with no sucrose in the
L. Spı
´
chal et al. PI-55 blocks cytokinin action
FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS 251
medium as described previously [24]. Twenty seeds for each
treatment were analyzed in three replicates.
Cytokinin bioassays
Standard bioassays based on stimulation of cytokinin-
dependent tobacco callus growth, the retention of chloro-
phyll in excised wheat leaves and the dark induction of
betacyanin synthesis in Amaranthus cotyledons were carried
out as described previously [20]. The only exception to the

published protocols was that the incubation time was short-
ened to 17 h in the Amaranthus bioassay. The final concen-
tration of dimethylsulfoxide in the media did not exceed
0.2%. Five replicates were prepared for each cytokinin con-
centration and the entire tests were repeated at least three
times.
Acknowledgements
We thank Jarmila Balonova
´
and Miloslava S
ˇ
ubova
´
for
skillful technical assistance; Dr Vladimı
´
r Krys
ˇ
tof for
testing of PI-55 cytotoxicity with human cell lines;
Katharina Achazi for help with introgression of the
ARR5:GUS marker gene into the ahk Arabidopsis
double mutant lines; and Professor David Morris for
his helpful suggestions and critical reading of the man-
uscript. This work was supported by the Grant Agency
of the Czech Republic (522 ⁄ 07 ⁄ P197 to L.S.); the
Czech Ministry of Education (MSM 6198959216 and
MSM LC06034 to M.S.); and the Deutsche Fors-
chungsgemeinschaft (Sfb 449 to T.S.).
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Supporting information
The following supplementary material is available:
Doc. S1. Synthesis, elemental and melting point analy-
sis, analytical thin layer chromatography, MS and
NMR analyses of PI-55.
Fig. S1. Effect of PI-55 on cytokinin binding to
AHK3.
Fig. S2. Comparison of PI-55 and BA effect of on pri-
mary root length of Arabidopsis wild-type seedlings.
Table S1. Effect of compound PI-55 on proliferation
of human cell lines and tobacco callus.
This supplementary material can be found in the

online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
L. Spı
´
chal et al. PI-55 blocks cytokinin action
FEBS Journal 276 (2009) 244–253 ª 2008 The Authors Journal compilation ª 2008 FEBS 253