Journal of Integrative Plant Biology 2010, 52 (1): 53–60

Invited Expert Review

Metabolism and Long-distance Translocation
of Cytokinins
∗
Toru Kudo, Takatoshi Kiba and Hitoshi Sakakibara
RIKEN Plant Science Center, Yokohama, Kanagawa, 230-0045, Japan
∗
Corresponding author
Tel: +81 45 503 9576; Fax: +81 45 503 9609; E-mail: sakaki@riken.jp
Available online on 6 January 2010 at www.jipb.net and www.interscience.wiley.com/journal/jipb
doi: 10.1111/j.1744-7909.2010.00898.x

Abstract

During plant development, distantly-located organs must commu-

Hitoshi Sakakibara
(Corresponding author)
nicate in order to adapt morphological and physiological features in
response to environmental inputs. Among the recognized signaling
molecules, a class of phytohormones known as the cytokinins
functions as both local and long-distance regulatory signals for
the coordination of plant development. This cytokinin-dependent
communication system consists of orchestrated regulation of the
metabolism, translocation, and signal transduction of this phyto-
hormone class. Here, to gain insight into this elaborate signal-
ing system, we summarize current models of biosynthesis, trans-
membrane transport, and long-distance translocation of cytokinins
in higher plants.

Kudo T, Kiba T, Sakakibara H (2010) Metabolism and long-distance translocation of cytokinins. J. Integr. Plant Biol. 52(1), 53–60.

Introduction
Higher plants are composed of multiple organ systems that
are functionally differentiated, such as photosynthetic and
non-photosynthetic organs and vegetative and reproductive
organs. Plant organs interact with each other to optimize both
metabolic and developmental processes to allow the organism
to accommodate to environmental inputs. For these mutual
interactions, local and long-distance communication among
cells and organs are essential. Messenger molecules, such as
phytohormones, mRNA, small RNAs and proteins, are involved
in this communication system and are transported throughout
the plant by the vascular system (Ruiz-Medrano et al. 2001;
Mouchel and Leyser 2007; Liu et al. 2009). Cytokinins, a class
of phytohormones, are one of these long-distance messengers
transported through the plant vascular system.
Cytokinins are defined as substances that induce cytoki-
nesis in the presence of auxin. To date, a variety of nat-
ural cytokinin species, including trans-zeatin (tZ), N 6 -(2 -
isopentenyl)adenine (iP), cis-zeatin (cZ), and their conjugates
have been identified; the active cytokinin species are the free-
base type (Mok and Mok 2001). In addition to their action
as inducers of cytokinesis, cytokinins are also involved in
regulating various biological processes: senescence (Gan and
Amasino 1995; Kim et al. 2006), apical dominance (Sachs
and Thimann 1967; Tanaka et al. 2006; Shimizu-Sato et al.
2009), root proliferation (Werner et al. 2001, 2003), phyllotaxis
(Giulini et al. 2004), and reproductive competence (Ashikari
et al. 2005). To regulate such plant developmental processes,
cytokinin activity must be finely controlled.
Cytokinin activity in an organ is regulated at diverse steps,
including de novo synthesis, activation, conjugation, and degra-
dation. Spatial distribution of cytokinin signaling systems (i.e.
receptors and response regulators) specifies the domain in
which a cytokinin response can occur. In addition, local and
long-distance transport systems are involved in regulating
cytokinin action. In the present review, we summarize the
role of cytokinins as a signaling messenger with focus on
the biosynthesis, transmembrane transport, and long-distance
translocation. For an update on intracellular cytokinin signal


C 2010 Institute of Botany, Chinese Academy of Sciences
54 Journal of Integrative Plant Biology Vol. 52
transduction, readers are directed to other excellent reviews
(Hwang et al. 2002; Heyl and Schmülling 2003; Kakimoto 2003;
Mizuno 2004; Argueso et al. 2009).
Cytokinin Metabolism in Higher Plants
In the past decade, our understanding of cytokinin biosynthesis
has greatly progressed due in large part to the identifica-
tion of key pathway genes encoding adenosine phosphate-
isopentenyltransferase (IPT; Kakimoto et al. 2001; Takei et al.
2001a; Sakamoto et al. 2006), tRNA-isopentenyltransferase
(tRNA-IPT; Miyawaki et al. 2004, 2006; Sakamoto et al. 2006),
cytokinin trans-hydroxylase, CYP735A (Takei et al. 2004b) and
the cytokinin nucleoside 5 -monophosphate phosphoribohydro-
lase, LONELY GUY (LOG; Kurakawa et al. 2007). Based on
these findings, a basic scheme for the cytokinin biosynthesis
pathway was proposed (Sakakibara 2006; Hirose et al. 2008;
Kamada-Nobusada and Sakakibara 2009) and is illustrated in
Figure 1.
In Arabidopsis, the initial step of iP and tZ biosynthesis is
catalyzed by IPT using dimethylallyl diphosphate (DMAPP) and
adenosine 5 -diphosphate (ADP), or adenosine 5 -triphosphate
Figure 1. Simplified model for cytokinin biosynthesis and
degradation pathways.
Blue arrows indicate reactions for enzymes with known genes, and
grey arrows indicate pathway genes that remain to be identified.
In this scheme, only biosynthesis and degradation steps are il-
lustrated; further details can be found in Sakakibara (2006). CKX,
cytokinin oxidase/dehydrogenase; cZ, cis-zeatin; DMAPP, dimethy-
lallyl diphosphate; iP, N 6 -(2 -isopentenyl)adenine; IPT, adenosine
phosphate-isopentenyltransferase; LOG, LONELY GUY; tRNA-IPT,
tRNA-isopentenyltransferase; tZ, trans-zeatin.
No. 1 2010
(ATP), to generate iP-ribotides. These iP-ribotides are then
hydroxylated to tZ-ribotides by CYP735A1 or CYP735A2 (Takei
et al. 2004b). On the other hand, biosynthesis of cZ is initiated
by tRNA-IPTs that catalyze the prenylation of tRNA using
DMAPP; however, the enzyme for cis-hydroxylation has yet
to be identified in plants. Conversion of iP-, tZ-, and cZ-
riboside 5 -monophosphate to their active forms occurs by two
pathways: the LOG and two-step pathways. In the former,
cytokinin riboside 5 -monophosphates are directly converted
to free-base cytokinins by LOG (Kurakawa et al. 2007). In
the latter pathway, the ribotides are dephosphorylated to the
ribosides and subsequently converted to free-base cytokinins
(Chen and Kristopeit 1981a, 1981b), but the corresponding
genes have not yet been identified.
Inactivation of cytokinins is carried out by degradation
or conjugation. Degradation is catalyzed by cytokinin oxi-
dase/dehydrogenase (CKX; Galuszka et al 2001; Schmülling
et al. 2003; Figure 1). Glucose-conjugation to cytokinins occurs
at the N3, N7 and N9 positions of the purine ring, or in the
hydroxyl group of the prenyl side chain. Recent studies have
demonstrated that the degradation step plays an important
role in regulating cytokinin activity (Werner et al. 2001, 2003;
Ashikari et al. 2005).
Traditionally it was thought that cytokinins were synthesized
in the root and transported to the shoots through the xylem
(Letham and Palni 1983; Beveridge et al. 1997). However,
recent studies on the spatial distribution of cytokinin metabolism
have demonstrated that cytokinins are produced not only
in roots, but also in various sites within the aerial parts of
the plant. In Arabidopsis, the IPT genes are expressed in
numerous organs including roots, leaves, stems, flowers, and
siliques (Miyawaki et al. 2004; Takei et al. 2004a), whereas
the CYP735A genes are expressed predominantly in roots
(Takei et al. 2004b). Recent studies on the LOG family genes
in rice and Arabidopsis suggest that activation of cytokinin
occurs in nearly all parts of the plant (Kurakawa et al. 2007;
Kuroha et al. 2009). Superimposition of the expression patterns
for the IPT and CYP735A genes, in Arabidopsis, reveals the
differential distribution of de novo synthesis pathways for iP and
tZ. For instance, AtIPT3 is expressed in phloem tissue in rosette
leaves, whereas expression of CYP735As in rosette leaves is
scarcely detectable. Alternatively, both IPTs and CYP735As
are expressed in roots. Such differential distribution of these
cytokinin biosynthesis genes might be important to produce
the various cytokinin species in underground and aboveground
organs.
Cytokinin Transport across
the Plasma Membrane
As cytokinins are a mobile class of phytohormones, it is
likely that higher plants have import and export systems to
 Cytokinin Metabolism and Translocation 55

mobilize the cytokinin across the plasma membrane (Cedzich

et al. 2008; Hirose et al. 2008). Characterization of cytokinin
transport in Arabidopsis cell cultures suggested the pres-
ence of proton-coupled multiphasic cytokinin transport systems
(Cedzich et al. 2008). To date, the purine permease (PUP)
family and the equilibrative nucleoside transporter (ENT) family
have been proposed as candidates for cytokinin transporters.
Among Arabidopsis PUP family proteins (Gillissen et al. 2000),
the ability of AtPUP1 and AtPUP2 to transport tZ and iP was
shown using a yeast system (Bürkle et al. 2003); however,
genetic studies on plant PUPs using loss-of-function or gain-
of-function mutants have not been reported.
For plant ENT proteins, competitive uptake studies in yeast
cells suggested that Arabidopsis ENT3, ENT6, ENT7 and rice
ENT2 can transport iP-riboside (iPR) and tZ-riboside (tZR)
(Hirose et al. 2005, 2008). Genetic screening for suppressor Figure 2. A possible model of cytokinin transport across the
mutants of cytokinin overproduction (Sun et al. 2003) resulted plasma membrane.
in the identification of T-DNA insertion lines in which AtENT8
Arabidopsis purine permease 1 (AtPUP1) and AtPUP2 can transport
expression was downregulated (Sun et al. 2005). Although
free-base cytokinins, such as iP and tZ, in a proton-coupled manner
these results suggest that plant ENT proteins are involved in the
(Gillissen et al. 2000; Bürkle et al. 2003). Some equilibrative
transport of cytokinin ribosides, clear and definitive evidence
nucleoside transporter (ENT) family proteins of Arabidopsis and rice
has not been provided. Figure 2 summarizes the manner in
can transport cytokinin ribosides such as iPR and tZR (Hirose et al.
which these putative cytokinin membrane transporters function.
2005, 2008; Sun et al. 2005). ENT family proteins, which have been
Clearly, further genetic studies on PUPs, ENTs, and other
identified also in mammals, fungi, and bacteria, facilitate diffusion
transporters are needed to fully understand such putative cy-
of nucleosides along a concentration gradient (Hyde et al. 2001).
tokinin transport systems establishing concentration gradients
iP, N 6 -(2 -isopentenyl)adenine; iPR, N 6 -(2 -isopentenyl)adenine
within specific tissues/organs.
riboside; tZ, trans-zeatin; tZR, trans-zeatin riboside.

Long-Distance Transport of Cytokinins
In higher plants, long-distance translocation of cytokinins is
mediated by the xylem, an acropetal transport system that
occurs by transpiration flow, and the phloem translocation
system that delivers photosynthate throughout the body of the
plant. Systemic translocation of cytokinins was implied by early
tracer experiments. Although radioactive cytokinins applied
to leaves are strongly retained at the treated site, a small
proportion of the labeled cytokinins are translocated to other
plant parts (Vonk and Davelaar 1981; Badenoch-Jones et al.
1984; Abo-hamed et al. 1984; Letham 1994). In xylem sap, the
major form of cytokinin is tZR (Beveridge et al. 1997; Takei et al.
2001b; Hirose et al. 2008), and in phloem sap, the major forms
are iP-type cytokinins, such as iPR and iP-ribotides (Corbesier
et al. 2003; Hirose et al. 2008). Thus, it is conceivable that
plants might use tZR as an acropetal messenger and iP-type
cytokinins as systemic or basipetal messengers (Figure 3).
This hypothesis is supported by a recent grafting experiment
using an atipt1;3;5;7 mutant, in which the content of both
iP-type and tZ-type cytokinins decreased in comparison with
wild-type plants (Miyawaki et al. 2006). Wild-type root-stocks
recovered the tZ-type cytokinins in the mutant shoot-scions
but not the iP-type cytokinins (Matsumoto-Kitano et al. 2008).
Wild-type shoot-scions recovered the iP-type cytokinins in the
mutant root-stocks to normal levels, whereas the tZ-type cy-
tokinins were only partially recovered (Matsumoto-Kitano et al.
2008). Reciprocal grafting experiments also restored visible
mutant phenotypes, such as defects in the thickening growth of
roots and inflorescence stems (Matsumoto-Kitano et al. 2008).
Summary of this grafting study is illustrated in Figure 4A and C.
Cytokinin translocation via the xylem is controlled both by
environmental and endogenous signals. The tZR content and
flow rate of the xylem sap are significantly increased by nitrate
supplement in barley (Samuelson et al. 1992) and maize
(Takei et al. 2001b), implying that tZR acts as a messenger
for nitrate signaling. Xylem cytokinins upregulated by nitrate
supplement induced the accumulation of cytokinin-responsive
gene transcripts in leaves (Sakakibara et al. 1998; Takei
et al. 2001b). In Arabidopsis roots, the accumulation of AtIPT3
transcripts is induced by nitrate, followed by that of tZ-ribotides
and tZR (Takei et al. 2002, 2004a) (Figure 3). Furthermore,
in an AtIPT3-deficient mutant, the nitrate-dependent accu-
mulation of cytokinins was markedly reduced or diminished
(Takei et al. 2004a), indicating that AtIPT3 is a key gene
for the nitrate-dependent de novo biosynthesis of cytokinins.
56 Journal of Integrative Plant Biology Vol. 52 No. 1 2010

Promoter-reporter analyses with transgenic Arabidopsis plants

showed that the AtIPT3 promoter is active in phloem compan-
ion cells rather than xylem tissues (Takei et al. 2004a). Thus,
there may well be a cytokinin translocation system operating
between the phloem and xylem tissues.
Studies on the increased-branching mutants of pea (rms4;
Beveridge et al. 1996) and Arabidopsis (max2; Stirnberg et al.
2002; Foo et al. 2007), in which the cytokinin content of
the xylem sap is dramatically reduced; imply a novel control
mechanism for cytokinin delivery via the xylem transpiration
stream. Reciprocal grafting experiments carried out between
rms4 and wild type plants showed that the reduction in xylem
cytokinin concentration and increase in branching occurs in
a scion-dependent manner (Beveridge et al. 1996). A scion-
dependent reduction in xylem cytokinin content was also ob-
served by reciprocal grafting experiments between max2 and
wild type plants (Foo et al. 2007). These results imply that some
endogenous and basipetal signals are involved in controlling
long-distance cytokinin movement. Summary of these grafting
studies are illustrated in Figure 4B and C. Currently, in addition
to cytokinin and auxin, strigolactone is proposed as a novel
phytohormone involved in branching (Gomez-Roldan et al.
2008; Umehara et al. 2008), but the identity of the signals
regulating xylem cytokinin levels remains to be elucidated.

Future Perspectives
Over the past decade, identification and characterization
of cytokinin-related genes has greatly advanced our under-
standing of cytokinin metabolism and signal transduction;
however, to fully elucidate the global signaling system for
Figure 3. A model for long-distance cytokinin transport cytokinins, the following issues need to be resolved. First,
through the plant vascular system. tZ-type and iP-type cytokinins are differentially distributed
in xylem and phloem tissues, implying that they transfer
In the xylem (pale red column) and phloem (pale blue column), tZR
different biological messages. At present, the physiological
and iP-type cytokinins are major transported species, respectively
meaning of side chain structural variation remains to be
(Corbesier et al. 2003; Hirose et al. 2008). Supplying nitrate to
solved. An important approach to answering this question
Arabidopsis roots induces expression of AtIPT3, a cytokinin biosyn-
is to use loss-of-function mutants of CYP735As, in which
thesis gene (Takei et al. 2004a) (indicated by circled red letters),
tZ-type cytokinin is expected to decrease or disappear. In
which subsequently upregulates the translocation of cytokinins (tZR)
addition, further characterization of ligand specificity and
through the xylem (red broken arrow). Cytokinin trans-hydroxylases
functional differentiation of cytokinin receptors will provide
(CYP735A1 and CYP735A2 in Arabidopsis) are involved in the
a basis to address this question. Second, it is now known
synthesis of tZR (Takei et al. 2004b). Xylem cytokinins (tZR) are
that nitrate acts as an environmental variable to control long-
translocated acropetally (red arrow) by the transpiration stream
distance cytokinin transport. However, additional factors are
(cyan arrow). Nitrate ions are also transported via xylem (black
likely to be involved in regulating this transport system. For
arrow) and assimilated into amino acids in the leaves. Phloem
instance, light conditions also affect cytokinin delivery to the
cytokinins (iP) are translocated systemically or basipetally (blue ar-
shoots by changing the transpiration rate (Boonman et al.
rows). Cytokinin biosynthesis and response (purple arrows) occur at
2007). Elucidation of mutual interactions between cytokinin
numerous sites throughout the plant. CK, cytokinin; iP-CK, N 6 -(2 -
delivery and these factors will be necessary in order to full
isopentenyl)adenine-type cytokinins; IPT, adenosine phosphate-
understand the physiological role of long-distance cytokinin
isopentenyltransferase; N, nitrogen; tZ, trans-zeatin; tZR, trans-
translocation for plant growth and development.
zeatin riboside.
 Cytokinin Metabolism and Translocation 57

Agricultural Implications
An increase of agricultural food production worldwide over the
past four decades has been associated with a remarkable in-
crease in the use of fertilizers (Hirel et al. 2007). Concomitantly,
this fertilizer usage also caused environmental concerns such
as the eutrophication of freshwater (London 2005) and marine
ecosystems (Beman et al. 2005). Since the world population
is growing, it will be essential over the foreseeable future
that increases in food production be achieved without further
negative impacts on the global environment. To meet this
important requirement, plant scientists will need to advance
our understanding of approaches that can be used to develop
crop plants with enhanced nutrient use efficiency. Plant produc-
tivity can be regulated by two factors: the morphological (e.g.
grain number) and metabolic (e.g. uptake and efficient use of
nutrients) attributes. Cytokinins are closely relevant to both of
these characteristics.

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

(A) An Arabidopsis mutant, atipt1;3;5;7, in which the content of both

iP-type (iP-CK) and tZ-type (tZ-CK) cytokinins is low, were recipro-
cally grafted with wild type plants (WT). WT root-stocks recovered
the tZ-CK in the mutant shoot-scions but not the iP-CK. WT shoot-
scions recovered the iP-CK in mutant root-stocks to normal levels,
whereas the tZ-CK level was only partially recovered. Small, middle,
and large ellipses around iP-CK or tZ-CK schematically indicate
each level of these cytokinins, representing decreased, partially
recovered and normal levels, respectively. Based on the work of
Matsumoto-Kitano et al. (2008).
(B) Increased-branching mutants of pea (rms4; Beveridge et al.
1996, 1997) and Arabidopsis (max2; Stirnberg et al. 2002; Foo
et al. 2007), in which xylem cytokinin content is decreased, were
reciprocally grafted with WT plants. Although the WT shoot-scion
could restore the xylem cytokinin content, the WT root-stock could
not. Cytokinin levels in xylem sap from root-stock are indicated with
thick (for the normal level) or narrow (for a low level) red arrows.
(C) Speculative models to explain the results of two grafting ex-
periments described in (A) (left side) and (B) (right side). The graft
experiments in (A) indicate that iP-CK can move basipetally but not
acropetally (blue arrow) and that tZ-CK can move acropetally (thick
orange arrow) rather than basipetally (narrow orange arrow). This
supports the hypothesis that plants might use tZR as an acropetal
messenger and iP-CK as basipetal messenger (Figure 3). The
partial recovery of tZ-CK in atipt1;3;5;7 root-stocks grafted to WT
scions is possibly caused by biosynthesis of tZ-CK from recovered
iP-CK, or basipetal translocation of tZ-CK. The latter result (B) is
Figure 4. Schematic illustrations of grafting studies used to
consistent with the hypothesis that some endogenous and basipetal
explore the long-distance translocation of cytokinins.
signals (broken green arrow) regulate xylem cytokinin translocation
(red arrow).
58 Journal of Integrative Plant Biology Vol. 52
In morphological aspects, a quantitative trait loci analysis
of rice grain number revealed that cytokinin activity is an
important factor to define grain number (Ashikari et al. 2005).
The loss-of-function mutations in the LOG gene caused severe
reduction in panicle size, abnormal branching patterns, and
a decrease in the number of flowers and stamens (Kurakawa
et al. 2007). In metabolic aspects, cytokinins are involved in the
regulation of various genes encoding transporters for nitrate,
ammonium, sulfate, phosphate, and iron (Sakakibara et al.
2006; Séguéla et al. 2008). Since such transporters mediate
primary uptake and proper allocation of essential nutrients, they
play an important role in efficient nutrient acquisition and usage.
Cytokinins also increase sink strength as initially demon-
strated by movement of radioactive metabolites, such as car-
bohydrates and amino acid, from other plant parts to cytokinin-
treated sites (Mothes et al. 1961; Mothes and Engelbrecht
1963; Kuiper 1993; Werner et al. 2008). In the rice grain, zeatin
content per grain undergoes an increase after heading and
the highest levels are present during the period of maximal
grain weight (Oritani and Yoshida 1976). Similarly in barley,
it was reported that cytokinins participate in the regulation
of grain size, possibly by influencing both accumulation and
the duration of the filling period (Mechael and Seiler-Kelbitsch
1972). These findings indicate that cytokinins are likely involved
in grain filling, a process that involves the active mobilization
of nutrients from a vascular system to the endosperm. Thus,
advancing our understanding of the regulatory network of
cytokinin signaling, metabolism, translocation, and action may
well provide a key to opening the door for further improvement
of agricultural outputs, including grain yield.
Acknowledgements
We thank Dr Norihito Nakamichi, RIKEN Plant Science Center,
Japan, for his helpful comments and critical reading of the
manuscript.
Received 20 Sept. 2009 Accepted 29 Oct. 2009
References
Abo-hamed S, Collin HA, Hardwick K (1984) Biochemical and phys-
iological aspects of leaf development in cocoa. VIII. Export and
distribution of 14
C auxin and kinin from the young and mature leaves.
New Phytol. 97, 219–225.
Argueso CT, Ferreira FJ, Kieber JJ (2009) Environmental perception
avenues: the interaction of cytokinin and environmental response
pathways. Plant Cell Environ. 32, 1147–1160.
Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T,
Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M (2005)
No. 1 2010
Cytokinin oxidase regulates rice grain production. Science 309,
741–745.
Badenoch-Jones J, Rolfe BG, Letham DS (1984) Phytohormones,
Rhizobium mutants, and nodulation in legumes. V. Cytokinin
metabolism in effective and ineffective pea root nodules. Plant
Physiol. 74, 239–246.
Beman JM, Arrigo K, Matson PM (2005) Agricultural runoff fuels large
phytoplankton blooms in vulnerable areas of the ocean. Nature 434,
211–214.
Beveridge CA, Murfet IC, Kerhoas L, Sotta B, Miginiac E, Rameau
C (1997) The shoot controls zeatin riboside export from pea roots.
Evidence from the branching mutant rms4. Plant J. 11, 339–
345.
Beveridge CA, Ross JJ, Murfet IC (1996) Branching in pea (action of
genes rms3 and rms4). Plant Physiol. 110, 859–865.
Boonman A, Prinsen E, Gilmer F, Schurr U, Peeters AJ, Voesenek
LA, Pons TL (2007) Cytokinin import rate as a signal for photo-
synthetic acclimation to canopy light gradients. Plant Physiol. 143,
1841–1852.
Bürkle L, Cedzich A, Döpke C, Stransky H, Okumoto S, Gillissen
B, Kühn C, Frommer WB (2003) Transport of cytokinins mediated
by purine transporters of the PUP family expressed in phloem,
hydathodes, and pollen of Arabidopsis. Plant J. 34, 13–26.
Cedzich A, Stransky H, Schulz B, Frommer WB (2008) Characteriza-
tion of cytokinin and adenine transport in Arabidopsis cell cultures.
Plant Physiol. 148, 1857–1867.
Chen CM, Kristopeit SM (1981a) Metabolism of cytokinin: dephospho-
rylation of cytokinin ribonucleotide by 5 -nucleotidases from wheat
germ cytosol. Plant Physiol. 67, 494–498.
Chen CM, Kristopeit SM (1981b) Metabolism of cytokinin: deribosyla-
tion of cytokinin ribonucleoside by adenine nucleosidase from wheat
germ cells. Plant Physiol. 68, 1020–1023.
Corbesier L, Prinsen E, Jacqmard A, Lejeune P, Van Onckelen
H, Périlleux C, Bernier G (2003) Cytokinin levels in leaves, leaf
exudate and shoot apical meristem of Arabidopsis thaliana during
floral transition. J. Exp. Bot. 54, 2511–2517.
Foo E, Morris SE, Parmenter K, Young N, Wang H, Jones A,
Rameau C, Turnbull CG, Beveridge CA (2007) Feedback regula-
tion of xylem cytokinin content is conserved in pea and Arabidopsis.
Plant Physiol. 143, 1418–1428.
Galuszka P, Frebort I, Sebela M, Sauer P, Jacobsen S, Pec P (2001)
Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin
degradation in cereals. Eur. J. Biochem. 268, 450–461.
Gan S, Amasino RM (1995) Inhibition of leaf senescence by autoreg-
ulated production of cytokinin. Science 270, 1986–1988.
Gillissen B, Bürkle L, André B, Kühn C, Rentsch D, Brandl B,
Frommer WB (2000) A new family of high-affinity transporters for
adenine, cytosine, and purine derivatives in Arabidopsis. Plant Cell
12, 291–300.
Giulini A, Wang J, Jackson D (2004) Control of phyllotaxy by the
cytokinin-inducible response regulator homologue ABPHYL1. Na-
ture 430, 1031–1034.

Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pagès V, Dun
EA, Pillot JP, Letisse F, Matusova R, Danoun S, Portais
JC, Bouwmeester H, Bécard G, Beveridge CA, Rameau C,
Rochange SF (2008) Strigolactone inhibition of shoot branching.
Nature 455, 189–194.
Heyl A, Schmülling T (2003) Cytokinin signal perception and trans-
duction. Curr. Opin. Plant Biol. 6, 480–488.
Hirel B, Le Gouis J, Ney B, Gallais A (2007) The challenge of
improving nitrogen use efficiency in crop plants: towards a more
central role for genetic variability and quantitative genetics within
integrated approaches. J. Exp. Bot. 58, 2369–2387.
Hirose N, Makita N, Yamaya T, Sakakibara H (2005) Functional char-
acterization and expression analysis of a gene, OsENT2, encoding
an equilibrative nucleoside transporter in rice suggest a function in
cytokinin transport. Plant Physiol. 138, 196–206.
Hirose N, Takei K, Kuroda T, Kamada-Nobusada T, Hayashi H,
Sakakibara H (2008) Regulation of cytokinin biosynthesis, com-
partmentalization and translocation. J. Exp. Bot. 59, 75–83.
Hwang I, Chen HC, Sheen J (2002) Two-component signal transduc-
tion pathways in Arabidopsis. Plant Physiol. 129, 500–515.
Hyde RJ, Cass CE, Young JD, Baldwin SA (2001) The ENT family
of eukaryote nucleoside and nucleobase transporters: recent ad-
vances in the investigation of structure/function relationships and
the identification of novel isoforms. Mol. Membr. Biol. 18, 53–63.
Kakimoto T (2001) Identification of plant cytokinin biosynthetic en-
zymes as dimethylallyl diphosphate: ATP/ADP isopentenyltrans-
ferases. Plant Cell Physiol. 42, 677–685.
Kakimoto T (2003) Perception and signal transduction of cytokinins.
Annu. Rev. Plant Biol. 54, 605–627.
Kamada-Nobusada T, Sakakibara H (2009) Molecular basis for cy-
tokinin biosynthesis. Phytochemistry 70, 444–449.
Kim HJ, Ryu H, Hong SH, Woo HR, Lim PO, Lee IC, Sheen J, Nam
HG, Hwang I (2006) Cytokinin-mediated control of leaf longevity by
AHK3 through phosphorylation of ARR2 in Arabidopsis. Proc. Natl.
Acad. Sci. USA 103, 814–819.
Kuiper D (1993) Sink strength: Established and regulated by plant
growth regulators. Plant Cell Environ. 16, 1025–1026.
Kurakawa T, Ueda N, Maekawa M, Kobayashi K, Kojima M, Nagato
Y, Sakakibara H, Kyozuka J (2007) Direct control of shoot meris-
tem activity by a cytokinin-activating enzyme. Nature 445, 652–
655.
Kuroha T, Tokunaga H, Kojima M, Ueda N, Ishida T, Nagawa S,
Fukuda H, Sugimoto K, Sakakibara H (2009) Functional analyses
of LONELY GUY cytokinin-activating enzymes reveal the impor-
tance of the direct activation pathway in Arabidopsis. Plant Cell doi.
10.1105/tpc.109.068676.
Letham DS (1994) Cytokinins as phytohormones – sites of biosynthe-
sis, translocation, and function of translocated cytokinin. In: Mok
DW, Mok MC, eds. Cytokinins: Chemistry, Activity, and Function.
CRC Press, Boca Raton, Florida. pp. 57–80.
Letham DS, Palni LMS (1983) The biosynthesis and metabolism of
cytokinins. Ann. Rev. Plant Physiol. 34, 163–197.
Cytokinin Metabolism and Translocation 59
Liu TY, Chang CY, Chiou TJ (2009) The long-distance signaling of
mineral macronutrients. Curr. Opin. Plant Biol. 12, 312–319.
London JG (2005) Nitrogen study fertilizes fears of pollution. Nature
433, 791.
Matsumoto-Kitano M, Kusumoto T, Tarkowski P, Kinoshita-
Tsujimura K, Václavı́ková K, Miyawaki K, Kakimoto T (2008)
Cytokinins are central regulators of cambial activity. Proc. Natl.
Acad. Sci. USA 105, 20027–20031.
Mechael G, Seiler-Kelbitsch H (1972) Cytokinin content and kernel
size of barley grain as affected by environmental and genetic factors.
Crop Sci. 12, 162–165.
Miyawaki K, Matsumoto-Kitano M, Kakimoto T (2004) Expression of
cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis:
tissue specificity and regulation by auxin, cytokinin, and nitrate.
Plant J. 37, 128–138.
Miyawaki K, Tarkowski P, Matsumoto-Kitano M, Kato T, Sato
S, Tarkowska D, Tabata S, Sandberg G, Kakimoto T (2006)
Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA
isopentenyltransferases in cytokinin biosynthesis. Proc. Natl. Acad.
Sci. USA 103, 16598–16603.
Mizuno T (2004) Plant response regulators implicated in signal trans-
duction and circadian rhythm. Curr. Opin. Plant Biol. 7, 499–505.
Mok DW, Mok MC (2001) Cytokinin metabolism and action. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 52, 89–118.
Mothes K, Engelbrecht L (1963) On the activity of a kinetin-like root
factor. Life Sci. 11, 852–857
Mothes K, Engelbrecht L, Schütte HR (1961) Über die Akkumulation
von a-Aminoisobuttersaüre im Blattgewebe unter dem Einfluss von
Kinetin. Physiol. Plant. 14, 72–75.
Mouchel CF, Leyser O (2007) Novel phytohormones involved in long-
range signaling. Curr. Opin. Plant Biol. 10, 473–476.
Oritani T, Yoshida R (1976) Studies on nitrogen metabolism in crop
plants. XIV. Changes in cytokinins in rice grains during ripening.
Pro. Crop Sci. Soc. Japan 45, 429–435.
Ruiz-Medrano R, Xoconostle-Cázares B, Lucas WJ (2001) The
phloem as a conduit for inter-organ communication. Curr. Opin.
Plant Biol. 4, 202–209.
Sachs T, Thimann KV (1967) The role of auxin and cytokinins in the
release of buds from dominance. Amer. J. Bot. 54, 134–144.
Sakakibara H (2006) Cytokinins: activity, biosynthesis, and transloca-
tion. Annu. Rev. Plant Biol. 57, 431–449.
Sakakibara H, Suzuki M, Takei K, Deji A, Taniguchi M, Sugiyama
T (1998) A response-regulator homologue possibly involved in
nitrogen signal transduction mediated by cytokinin in maize. Plant
J. 14, 337–344.
Sakamoto T, Sakakibara H, Kojima M, Nagasaki H, Yamamoto
Y, Inukai Y, Sato Y, Matsuoka M (2006) Ectopic expression of
KNOTTED1-like homeobox protein induces expression of cytokinin
biosynthesis genes in rice. Plant Physiol. 142, 54–62.
Samuelson ME, Eliasson L, Larsson CM (1992) Nitrate-regulated
growth and cytokinin responses in seminal roots of barley. Plant
Physiol. 98, 309–315.
60 Journal of Integrative Plant Biology Vol. 52 No. 1
Schmülling T, Werner T, Riefler M, Krupková E, Manns IB (2003)
Structure and function of cytokinin oxidase/dehydrogenase genes
of maize, rice, Arabidopsis and other species. J. Plant Res. 116,
241–252.
Séguéla M, Briat JF, Vert G, Curie C (2008) Cytokinins negatively
regulate the root iron uptake machinery in Arabidopsis through a
growth-dependent pathway. Plant J. 55, 289–300.
Shimizu-Sato S, Tanaka M, Mori H (2009) Auxin-cytokinin interactions
in the control of shoot branching. Plant Mol. Biol. 69, 429–435.
Stirnberg P, van De Sande K, Leyser HM (2002) MAX1 and MAX2
control shoot lateral branching in Arabidopsis. Development 129,
1131–1141.
Sun J, Niu QW, Tarkowski P, Zheng B, Tarkowska D, Sandberg
G, Chua NH, Zuo J (2003) The Arabidopsis AtIPT8/PGA22 gene
encodes an isopentenyl transferase that is involved in de novo
cytokinin biosynthesis. Plant Physiol. 131, 167–176.
Sun J, Hirose N, Wang X, Wen P, Xue L, Sakakibara H, Zuo J
(2005) Arabidopsis SOI33/AtENT8 gene encodes a putative equili-
brative nucleoside transporter that is involved in cytokinin transport
in planta. J. Integr. Plant Biol. 47, 588–603.
Takei K, Sakakibara H, Sugiyama T (2001a) Identification of genes
encoding adenylate isopentenyltransferase, a cytokinin biosynthe-
sis enzyme, in Arabidopsis thaliana. J. Biol. Chem. 276, 26405–
26410.
Takei K, Sakakibara H, Taniguchi M, Sugiyama T (2001b) Nitrogen-
dependent accumulation of cytokinins in root and the translocation
to leaf: implication of cytokinin species that induces gene expression
of maize response regulator. Plant Cell Physiol. 42, 85–93.
Takei K, Takahashi T, Sugiyama T, Yamaya T, Sakakibara H (2002)
Multiple routes communicating nitrogen availability from roots to
shoots: a signal transduction pathway mediated by cytokinin.
J. Exp. Bot. 53, 971–977.
2010
Takei K, Ueda N, Aoki K, Kuromori T, Hirayama T, Shinozaki K,
Yamaya T, Sakakibara H (2004a) AtIPT3 is a key determinant of
nitrate-dependent cytokinin biosynthesis in Arabidopsis. Plant Cell
Physiol. 45, 1053–1062.
Takei K, Yamaya T, Sakakibara H (2004b) Arabidopsis CYP735A1
and CYP735A2 encode cytokinin hydroxylases that catalyze the
biosynthesis of trans-zeatin. J. Biol. Chem. 279, 41866–41872.
Tanaka M, Takei K, Kojima M, Sakakibara H, Mori H (2006) Auxin
controls local cytokinin biosynthesis in the nodal stem in apical
dominance. Plant J. 45, 1028–1036.
Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-
Kamiya N, Magome H, Kamiya Y, Shirasu K, Yoneyama K,
Kyozuka J, Yamaguchi S (2008) Inhibition of shoot branching by
new terpenoid plant hormones. Nature 455, 195–200.
Vonk CR, Davelaar E (1981) 8–14 C-Zeatin metabolites and their
transport from leaf to phloem exudates of Yucca. Physiol. Plant.
52, 101–107.
Werner T, Holst K, Pörs Y, Guivarćh A, Mustroph A, Chriqui D,
Grimm B, Schmülling T (2008) Cytokinin deficiency causes distinct
changes of sink and source parameters in tobacco shoots and roots.
J. Exp. Bot. 59, 2659–2672.
Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H,
Schmülling T (2003) Cytokinin-deficient transgenic Arabidopsis
plants show multiple developmental alterations indicating opposite
functions of cytokinins in the regulation of shoot and root meristem
activity. Plant Cell 15, 2532–2550.
Werner T, Motyka V, Strnad M, Schmülling T (2001) Regulation of
plant growth by cytokinin. Proc. Natl. Acad. Sci. USA 98, 10487–
10492.
(Co-Editor: William J. Lucas)