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The role of cytokinins in root development of pea seedlings

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DOI: 10.1023/A:1013314919299

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Planta (2008) 227:1279–1289
DOI 10.1007/s00425-008-0699-z

ORIGINAL ARTICLE

Spatial and temporal changes in endogenous cytokinins

in developing pea roots
W. A. Stirk Æ O. Novák Æ K. Václavı́ková Æ
P. Tarkowski Æ M. Strnad Æ J. van Staden

Received: 10 October 2007 / Accepted: 16 January 2008 / Published online: 13 February 2008
Ó Springer-Verlag 2008

Abstract Germination and seedling establishment follows cytokinins were detected in the root tip, whereas trans-
a distinct pattern which is partly controlled by hormones. zeatin- (tZ), dihyrozeatin- (DHZ) and iP-type cytokinins
Roots have high levels of cytokinins. By quantifying the were found in the secondary roots and root zone. Cytokinin
fluctuations in endogenous cytokinins over time, further biosynthesis was only detected after day 6. Biosynthesis of
insight may be gained into the role of cytokinins during iP and tZ derivatives was quite rapid, whereas biosynthesis
germination and seedling establishment. Radicles were of cZ derivatives remained at a low basal level. These
excised from sterile Pisum sativum L. seeds after 30 min and fluctuations in cytokinin types and concentrations suggest
5 h imbibition. Seedlings germinated on agar were har- the cytokinins may be synthesized from various pathways in
vested after 1, 3, 6 and 9 days. The roots were divided into pea roots.
the root tip, root free zone, secondary root zone and from day
6, the secondary roots. Samples were purified by various
Keywords Cytokinins
Germination
Pisum

chromatographic methods and endogenous cytokinins
Root tip
Secondary roots
Seedling establishment
detected by LC(+)ES-MS. Benzyladenine levels doubled
after 5 h imbibition and then gradually decreased over time.
Low concentrations of cis-Zeatin (cZ) type cytokinins were Abbreviations
detected in the radicle after 30 min imbibition. After 5 h BA N6-benzyladenine
imbibition, cis-zeatin riboside-50 -monophosphate had CBP Cytokinin binding proteins
greatly increased. The total cytokinin content of the roots cZ cis-Zeatin
increased over time with the ribotides being the predominant cZOG cis-Zeatin-O-glucoside
conjugates. From day 3 onwards, there was a gradual cZR cis-Zeatin riboside
increase in the free bases, O-glucosides and their ribosylated cZROG cis-Zeatin riboside-O-glucoside
forms. Mainly N6-(2-isopentenyl)adenine (iP)-type cZRMP cis-Zeatin riboside-50 -monophosphate
DEAE Diethylaminoethyl
DHZ Dihydrozeatin
DHZRMP Dihydrozeatin riboside-50 -monophosphate
IAC Immunoaffinity chromatography
W. A. Stirk (&)
J. van Staden
iP N6-(2-isopentenyl)adenine
Research Centre for Plant Growth and Development, iPRMP N6-(2-isopentenyl)adenosine-
School of Biological and Conservation Sciences, 50 -monophosphate
University of KwaZulu-Natal Pietermaritzburg, IPT Isopentenyltransferase
Private Bag X01, Scottsville 3209, South Africa
e-mail: stirk@ukzn.ac.za
LC(+)ES-MS Liquid chromatography with electrospray
interface mass spectrometry
O. Novák
K. Václavı́ková
P. Tarkowski
M. Strnad MS Mass spectrometry
Laboratory of Growth Regulators, Palacký University & Institute mT meta-Topolin
of Experimental Botany AS CR, Šlechtitelů 11,
783 71 Olomouc, Czech Republic
tRNA Transfer ribonucleic acid

123
1280
tZ trans-Zeatin
tZR trans-Zeatin riboside
tZRMP trans-Zeatin riboside-50 -monophosphate
Introduction
Germination and development of seedlings follows a series
of overlapping morphological and physiological events that
are often triggered by imbibition. Such events include
radicle emergence, secondary root initiation and elongation
and shoot establishment. These events are tightly regulated
by a number of physiological factors, with each develop-
mental phase having different requirements. Pea seedlings
have been extensively studied with respect to their germi-
nation and secondary root development. In intact pea
seedlings, secondary roots initiated from meristems within
the root, form a distinct band called the root zone. The root
zone is separated from the root tip by a root free zone. Thus
there is spatial separation between the dividing cells in the
root tip, elongating cells in the root free zone and the
secondary root meristem. The establishment of this pattern
is, in part, determined by plant hormone concentrations
(Hinchee and Rost 1986). A gradient of endogenous
cytokinins was found in 3-day-old pea roots with the
cytokinin concentration being far greater in the meriste-
matic root tip compared to the segment immediately behind
it (Short and Torrey 1972). This gradient is consistent with
the gene expression results for Arabidopsis roots (Lohar
et al. 2004).
Although the sites of cytokinin biosynthesis are not
well defined, tissues with high-cytokinin concentrations
are presumed to be sites of biosynthesis (Miyawaki et al.
2004). Roots are reported to be the main site of cyto-
kinin biosynthesis although there is now evidence that
other tissues with the capacity for cell division, such as
young leaves, shoot apical meristems and immature
seeds, can also synthesize cytokinins (Miyawaki et al.
2004; Nordström et al. 2004a).
Three groups of cytokinins are recognized based on their
side chain nature: (1) the isoprenoid cis-zeatin (cZ), trans-
zeatin (tZ) and N6-(2-isopentenyl)adenine (iP), (ii) iso-
prenoid-derived dihydrozeatin (DHZ) and (iii) the aromatic
N6-benzyladenine (BA) and topolins (Zažı́malová et al.
1999). These are present in the plant in various conjugate
forms (Haberer and Kieber 2002). Local cytokinin levels
are controlled by the balance of biosynthesis, intercon-
version, transport and degradation (Miyawaki et al. 2004).
Metabolic conversion between the various cytokinins is
achieved by a number of enzymes, some of which have
been isolated and characterized (Sakakibara 2006). Even
small substitutions in the cytokinin side-chain have a
123
Planta (2008) 227:1279–1289
pronounced effect on their sensitivity to specific cytokinin
receptors and their biological activity (Spı́chal et al. 2004;
Sakakibara 2006).
The contribution of individual cytokinins to the various
growth processes is not clearly understood, with each
conjugate type potentially having a different role. Cytoki-
nin-free bases and ribosides are considered the most
biologically active cytokinin forms but are susceptible to
irreversible degradation by cytokinin oxidases (Haberer
and Kieber 2002; Spı́chal et al. 2004; Sakakibara 2006). As
O-glucosyl conjugates are resistant to cytokinin oxidases
and readily converted into the active free bases and ribo-
sides by the highly substrate specific glucosidases, they are
considered as inactive but stable forms of excess cytokinins
that play an important role in balancing cytokinin levels
(Spı́chal et al. 2004; Sakakibara 2006). The adenine ring
structure can also be glucosylated at the N3-, N7- and N9-
position. As these conjugates are usually inactive in bio-
assays and not reversibly sequestrated, their in vivo
function is believed to be deactivation (Haberer and Kieber
2002).
It is important to consider the types and concentra-
tions of endogenous cytokinins present in plant tissue at
any given time to gain a better understanding of the role
of specific cytokinins in the various developmental pro-
cesses. The spatial and temporal distribution of
biologically active cytokinins is tightly regulated in
plants to co-ordinate specific developmental events
(Kurakawa et al. 2007). Growth and development in
plants is a balance between cell division and differenti-
ation (Kyozuka 2007). Cytokinins are one of the key
hormones in root development, having a negative regu-
latory function (Werner et al. 2003). Cytokinins promote
cell division, vascular cambium sensitivity and differen-
tiation apical dominance in roots (Aloni et al. 2006).
They work antagonistically with other hormones, espe-
cially auxin. Cytokinin-deficient transgenic Arabidopsis
plants have increased root branching (Werner et al.
2003) and enlarged root meristems, with cells undergo-
ing more divisions before exiting the meristem and
undergoing elongation (Werner et al. 2001). This cyto-
kinin action is localized in the vascular tissue at the
transition zone in the root meristem where it acts as an
antagonist to auxin (Ioio et al. 2007).
Recent advances performed in analytical techniques
now allow for the accurate quantification of very low
concentrations of the hormones. The aim of this study was
to identify and quantify the endogenous cytokinins found
in the various components of intact pea seedling roots as
they develop over time. This study may provide insight
into the role of the various cytokinins during germination
and seedling establishment.
Planta (2008) 227:1279–1289
Materials and methods
Plant material and growth conditions
One sample of 200 radicles was prepared by soaking pea
seeds (Pisum sativum L. var. Greenfeast, Starke Ayres,
South Africa) in distilled water for 30 min to soften the
testa sufficiently so that the ‘‘dry’’ radicles could be
excised. Another sample of 200 seeds was also prepared
where the radicles were excised from seeds that had been
imbibed for 5 h.
Additional pea seeds were soaked in 3.5% NaOCl for
30 min and then rinsed well with sterilized water. The
surface sterilized seeds were partially submerged (to
reduce oxidative stress) in sterilized water for 5 h. The
seeds were then placed in tissue culture jars containing
0.5 g No. 3 Oxoid Bacteriological Agar dissolved in
100 ml distilled water as a support medium. There were
seven seeds per jar. All work was carried out on a laminar
flow bench to minimize contamination. The seeds were
grown in the dark at 22°C. After 1, 3, 6 and 9 days, 200
seedlings were harvested, and the root length and number
of secondary roots recorded. No seedlings from contami-
nated jars were used. Each seedling was then divided into a
2 mm root tip, the root free zone, the root zone and if
emerged, the secondary roots (days 6 and 9) as described in
the Introduction.
Identification and quantification of endogenous
cytokinins
The procedure used for cytokinin analysis was a modifica-
tion of the method described by Novák et al. (2003). Freeze-
dried root samples were extracted in triplicate for 3 h in ice-
cold 70% ethanol (v/v) and deuterium labelled standards
added, each at 5 pmol per sample to check recovery during
purification and to validate determination. The standards
were [2H5]tZ, [13C5]cZ, [2H5]tZR, [2H5]tZ9G, [2H5]tZRMP,
[2H3]DHZ, [2H3]DHZR, [2H3]DHZ9G, [2H3]DHZRMP
[2H6]iP, [2H6]iPR, [2H6]iP9G, [2H6]iPRMP, [2H5]tZOG,
[2H5]tZROG, [2H7]DHZOG, [2H7]DHZROG. The aromatic
cytokinins were analysed using the following internal stan-
dards: [2H7]BA, [2H7]BAR, [2H7]BA9G, [2H7]BARMP,
[15N4]mT, [15N4]oT, and [15N4]Kin (Olchemim Ltd., Czech
Republic).
After extraction, the homogenate was centrifuged
(15,000g, 4°C) and the pellets re-extracted. The combined
supernatants were concentrated to approximately 1.0 ml
under vacuum at 35°C, diluted to 20 ml with ammonium
acetate buffer (40 mM, pH 6.5) and purified using a
combined DEAE-Sephadex (1.0 cm9 5.0 cm)-octadecyl-
silica (0.5 cm9 1.5 cm) column and immunoaffinity
1281
chromatography (IAC) based on generic cytokinin mono-
clonal antibody (Faiss et al. 1997). This solution resulted in
three fractions containing (1) the free bases, ribosides and
N-glucosides, (2) a ribotide fraction, and (3) an O-glucoside
fraction. Fractions 2 and 3 were also purified using IAC
where fraction 2 was treated with alkaline phosphatase
(Roche Diagnostics, Mannheim, Germany) and fraction 3
with b-glucosidase (G-0395, Sigma, St. Louis, USA).
The fractions from the IAC columns were evaporated to
dryness and dissolved in 50 ll of the mobile phase used for
HPLC-MS analysis. The samples were analysed by HPLC
(Waters Alliance 2690) linked to a Micromass ZMD 2000
single quadrupole mass spectrometer equipped with an
electrospray interface [LC(+)ES-MS] and diode-array
detector (Waters PDA 996). Using a post-column split of
1:1, the effluent was introduced into an electrospray source
(source block temperature 100°C, desolvation temperature
250°C, capillary voltage +3.0 kV, cone voltage 20 V) and
PDA (scanning range 210–300 nm; with 1.2 nm resolu-
tion), and quantitative analysis of the different cytokinins
was performed in a selective ion-monitoring mode.
Quantification was performed by Masslynx software using
a standard isotope dilution method. The ratio of endoge-
nous cytokinin to appropriate labelled standard was
determined and further used to quantify the level of
endogenous compounds in the original extract, according
to the knowledge of quantity of added internal standard
(Novák et al. 2003).
Cytokinin identification by exact mass determination
A CapLCÒ module (Waters, Milford, MA, USA) capillary
liquid chromatography system equipped with a reversed-
phase (Symmetry C18, 0.3 mm 9 150 mm, 5 lm, Waters)
column coupled to a hybrid Q-Tof micro mass analyzer
(Waters MS Technologies, Manchester, UK) was used for
high-resolution identification of cytokinin derivatives.
Following injection, cytokinins were eluted with a 25-min
binary linear gradient, again composed of 15 mM ammo-
nium formate (pH 4.0, A) and methanol (B) starting at an
A:B ratio of 1:9 (v/v), and finishing at a 1:1 ratio of A:B,
but with a flow-rate of 5 lL/min and column temperature
of 35°C. Electrospray ionization in the positive ion mode
was performed using the following parameters: source
block/desolvation temperature, 90/200°C; capillary/cone
voltage, 2,500/30 V; and spray/cone gas flow (N2),
50/250 L/h. In the full-scan mode, data were acquired in
the mass range 50–500 Da, with a cycle time of 28 ls, a
scan time of 1.0 s and a collision energy of 4 V. For the
MS/MS experiments, analytes were fragmented with the
collision cell filled with argon gas and collision energies of
15, 20 and 25 V. For the exact mass determination
123
1282
experiments, a lock spray was used for external calibration
with a mixture of 0.1 M NaOH/10% (v/v), formic acid and
acetonitrile (1:1:8, by vol) as a reference. Accurate masses
were calculated and used for the determination of the
elementary composition and structure of the analytes with
fidelity C5 ppm. All data were processed by the Quan-
LynxTM program included in the MassLynxTM software
package (version 4.0, Waters).
Measuring biosynthetic rates of cytokinin ribotides
and ribosides
Approximately 20 g of pea seeds were incubated in 100 ml
30% deuterium oxide at 23°C in a 16:8 h light:dark regime.
After 24 and 48 h incubation, seeds were gently dried
between sheets of filter paper and immediately frozen in
liquid nitrogen. Pea seedlings were grown on moist perlite
at 23°C in a 16:8 h light:dark regime. The roots of 6- and
9-day-old seedlings were dissected out and incubated with
30% deuterium oxide for 24 h and collected under the
same conditions. All samples were kept at -80°C until
extraction and purification.
Samples were extracted, purified and derivatized as
described by Åstot et al. (1998). Biosynthetic rates (t/t
ratio) of cytokinin ribotides and ribosides were determined
by the modified method described by Nordström et al.
(2004b) where 3 g fresh mass was extracted in 10 ml
Bieleski’s extraction solvent (Bieleski 1964) overnight at
-20°C. Extracts were passed in sequence through SCX
and DEAE-Sephadex-C18 cartridges. Fractions containing
the cytokinin ribosides were further purified by cytokinin-
specific IAC (Olchemim Ltd.). Fractions containing
cytokinin ribotides were treated with alkaline phosphatase
and subsequently also purified by the IAC. The dried
samples were propionylated using N,N-dimethylformam-
ide-N-methylimidazole-propionic anhydride (5:3:1 v/v/v),
then evaporated to dryness and stored at -20°C until
analysed.
The isotopomer profile was determined in multiple
reaction monitoring mode by UPLC (Waters Acquity)
linked to a Quatro Micro triple quadrupole mass spec-
trometer equipped with an electrospray interface
[LC(+)ES-MS/MS]. The incorporation of deuterium to
molecules of newly synthesized cytokinins was calculated
as a tracer/tracee ratio (t/t ratio) as outlined by Åstot et al.
(2000a). The t/t ratio was obtained by calculating the nat-
ural isotope distribution in a set of standards, then
subtracting the natural isotope contribution of each iso-
topomer in the sample by multiplying the natural isotope
fraction found in the standards by the [M+H]+ isotope and
subtracting the resulting value from the respective isotop-
omers. There were four replicates per treatment.
123
Planta (2008) 227:1279–1289
Results
The root length and number of secondary roots are shown
in Table 1. By day 1, the hypocotyl had emerged and it
continued to elongate throughout the experiment. A few
secondary roots were visible by day 6 and these had
increased in number and length by day 9.
Eighteen isoprenoid cytokinins were identified in the
radicle and seedling roots. The identity of all cytokinin
metabolites was verified by comparison of the mass spectra
and chromatographic retention times with those of
authentic standards. These consisted of the free bases tZ,
cZ, DHZ and iP and their corresponding riboside, riboside-
O-glucoside, O-glucoside and ribotide (50 -monophos-
phates) conjugates (Figs. 1, 3, 4, 5). No N-glucosides were
found. Only two aromatic cytokinins, BA and meta-topolin
(mT) were detected (Fig. 1e, f).
Very low concentrations or no isoprenoid-free bases
were detected in the radicles at day 0, even after 5 h
imbibition. The contribution of the free bases to the cyto-
kinin pool only increased slightly over time with increases
in tZ and iP (Fig. 1a, d). iP concentrations increased on day
3 with the highest concentration measured in the root tip.
High iP concentrations were also measured in the emerging
secondary roots on day 6 (Fig. 1d). Trans-zeatin concen-
trations increased on day 6, being detected mainly in the
emerging secondary roots and remained low in the root tip
(Fig. 1a). Concentrations of cZ and DHZ remained low
throughout the experiment (Fig. 1b, c) although cZ con-
centrations increased slightly after 5 h imbibition on day 0.
However, the radicles had a very high concentration of the
aromatic cytokinin BA. This concentration more than
doubled after 5 h imbibition and then decreased gradually
over time until only low concentrations were detected in
the root tip of the seedlings on day 9. BA was not detected
in the secondary roots (Fig. 1e). mT was the only topolin
detected in the pea roots and occurred in very low con-
centrations throughout the experiment (Fig. 1f).
The presence of high concentrations of aromatic cyto-
kinins (BA, mT) is also observed in many other plant
Table 1 Radicle length, root length and number of secondary roots
of the pea seedlings determined as the mean ± SD (n = 200)
Treatment Root length (mm) No. of secondary roots
30 min imbibition 3.0 ± 0.6a –
5 h imbibition 3.5 ± 0.6 a
–
Day 1 4.7 ± 0.7 0±0
Day 3 22.6 ± 6.5 0±0
Day 6 69.0 ± 18.5 4.55 ± 3.95
Day 9 98.0 ± 31.6 31.60 ± 10.31
a
Radicle
Planta (2008) 227:1279–1289 1283

Fig. 1 Changes in
a) t Z b) cZ
concentrations of cytokinin-free 50
bases trans-zeatin (tZ, a), cis-
zeatin (cZ, b), dihydrozeatin Root tip
40 Root free zone
(DHZ, c),
Root zone
N6-(2-isopentenyl)adenine (iP, 30 Secondary roots
d), N6-benzyladenine (BA, e)
and meta-topolin (mT, f) in pea 20
seedlings measured over 9 days
of incubation. LOD limit of Radicle
10
detection, error bars represent

<LOD
Radicle

CY TOKININ CONCENTRATION (pmol g-1 DW)

SD (n = 3)
0

c) DHZ d) iP
50

40

30

20

Radicle
10
<LOD
<LOD

<L OD

< LOD

<L OD
<L OD
<L OD
< L OD
< LO D
< LOD
Radicle

0
e)BA f) mT
197.3
50 231.4
88.6
79.6
40

30

20

10 Radicle
<LOD
<LOD

<LOD

<LOD
h <LOD

<LOD

<LOD
<LOD
<LOD
< LO D
0
1 3 6 9 3
in

1 6 9
in
h

m
m
5

5
30
30

TIME (days) TIME (days)

species during germination (own unpublished data) and concentration was trans-zeatin riboside (tZR). The con-
therefore appears to be of general importance during this centration of tZR remained very low in the root tip over the
physiological process. To confirm the real presence of 9 days but increased to high levels in the root-free zone and
cytokinins in germinating pea seeds, the identity of BA was root zone from day 3 onwards and the secondary roots from
verified by comparison of the mass spectra obtained by day 6 onwards (Fig. 3a). Although occurring in lower
Q-Tof MS and chromatographic retention with the syn- concentrations, the distribution of dihydrozeatin riboside
thetic standard. To further verify the presence of BA-like (DHZR) was similar to that of tZR (Fig. 1c). Conversely,
substance in the plant material, the tissue extracts were highest concentrations of cis-zeatin riboside (cZR) were
combined, immunopurified by BA-specific IACs (data not detected in the root tip and the secondary roots on day 6
shown), fractionated by HPLC and the corresponding UV (Fig. 3b). The highest N6-(2-isopentenyl)adenosine (iPR)
fractions collected and combined. The structure of almost concentration was measured in the secondary roots on day
pure BA obtained in this way was determined by Q-Tof 6 (Fig. 3d).
MS which can provide both structural elucidation and Very low concentrations of O-glucosides and riboside-
highly accurate mass determinations. The mass spectra of O-glucosides were present in the radicles on day 0. The
natural BA unequivocally identified in germinating pea O-glucosides did not increase over time while there was
seeds is shown in Fig. 2. a general increase in the riboside-O-glucoside concen-
All the riboside conjugates increased over time with trations, especially trans-zeatin riboside-O-glucoside
the highest concentrations generally being detected in the (tZROG) and cis-zeatin riboside-O-glucoside (cZROG)
tissues on day 6. The riboside found in the highest and to a lesser extent dihydrozeatin riboside-O-glucoside

123
1284 Planta (2008) 227:1279–1289

a) The ribotides occurred in the highest concentrations of

all the cytokinin types with a general increase over time.
b) These were the first cytokinin forms to be detected with
high levels of cZRMP measured 5 h after imbibition
(Fig. 5b), isopenteyladenosine-50 -monophosphate (iPRMP)
detected on day 1 (Fig. 5d) and trans-zeatin riboside-
50 -monophosphate (tZRMP) and dihyrozeatin riboside-
5-monophosphate (DHZRMP) measured on day 3 (Fig. 5a,
c). By day 9, high concentrations of cZRMP and iPRMP
were detected in the root tip although the highest concen-
trations were recorded in the root free zone (Fig. 5b, d).
Their distribution within the root was similar to the other
cytokinin conjugates, with the root tip having the highest
cZRMP and iPRMP concentrations and tZRMP and
DHZRMP being restricted mainly to the root zone. On day
6, the elongating secondary roots also contained high
concentrations of cZRMP (Fig. 5b).
Within the first 48 h following imbibition, no cytokinin
biosynthesis was detected in the pea seeds. After day 6,
biosynthesis of iP and tZ derivatives became quite rapid,
Fig. 2 a, b Identification of N6-benzyladenine (BA) in immunopu- whereas the biosynthesis of cZ derivatives remained at a
rified samples in germinating pea seeds detected by capillary liquid low basal level (Table 2).
chromatography-Q-Tof mass spectrometry (MS). Accurate masses
were calculated and used for the determination of the compound’s
elementary composition. a MS chromatogram (m/z 226) and b MS
spectrum of BA obtained from the basic fraction Discussion

(DHZROG; Fig. 4b, d, f). Their distribution within the Germination is defined as a triphasic event beginning with
root mirrored that of their corresponding riboside con- the uptake of water by quiescent dry seeds, followed by a
jugates (Fig. 4a, c, e). lag phase and ending with the elongation of the embryonic

Fig. 3 Changes in
100 a) tZR b) cZR
concentrations of cytokinin 121.0 201.4
ribosides trans-zeatin riboside Root tip
104
(tZR, a), cis-zeatin riboside 80 Root free zone
(cZR, b), dihydrozeatin riboside Root zone
CYTOKININ CONCENTRATION (pmol g-1 DW)

(DHZR, c) N6-(2- 60 Secondary roots

isopentenyl)adenosine (iPR, d )
in pea seedlings measured over 40
9 days of incubation. No
aromatic cytokinin ribosides 20
Radicle
were detected. LOD limit of
<LOD
<LOD

Radicle
detection, error bars represent
0
SD (n = 3)
100 c) DHZR d) iPR

80

60

40

20 Radicle
<LOD
<LOD
h <LOD

Radicle

0
1 3 6 9 1 3 6 9
in

in
h
m

m
5

5
30

30

TIME (days) TIME (days)

123
Planta (2008) 227:1279–1289 1285

Fig. 4 Changes in
concentration of cytokinin a) t ZOG b) t ZROG
50
O-glucosides and riboside-
Root tip
O-glucosides trans-zeatin- 40 Root free zone
O-glucoside (tZOG, a),
Root zone
trans-zeatin riboside- 30 Secondary roots
O-glucoside (tZROG, b), cis-
zeatin-O-glucoside (cZOG, c), 20
cis-zeatin riboside-O-glucoside
(cZROG, d), dihydrozeatin- 10 Radicle Radicle
O-glucoside (DHZOG, e)

<LOD
<LOD
<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD
CYTOKININ CONCENTRATION (pmol g-1 DW)
dihydrozeatin riboside-
0
O-glucoside (DHZROG, f) in
pea seedlings measured over c) cZOG d) cZROG
9 days of incubation. No 50
aromatic cytokinin conjugates
were detected. LOD limit of 40
detection, error bars represent
SD (n = 3) 30

20

Radicle Radicle
10
<LOD

<LOD
<LOD
0

e) DHZOG f) DHZROG
50

40

30

20

Radicle
10
<LOD

<LOD

<LOD
<LOD
<LOD

<LOD
<LOD

<LOD

<LOD

<LOD
<LOD
<LOD
<LOD

<LOD

<LOD
<LOD
<LOD

<LOD

<LOD
Radicle

0
1 3 6 9 1 3 6 9
in
in
h

h
m
m
5

5
30
30

TIME (days) TIME (days)

axis (Bewley and Black 1982; Bewley 1997). During the predominant zeatin isomers e.g. chickpea seeds, rice, maize
lag phase there is enzyme activation, de novo enzyme and white lupins (see Emery et al. 1998; Martin et al. 2001;
synthesis and the resumption of metabolic activities such as Veach et al. 2003). The results of the present study showed
respiration and nutrient mobilization (Bewley 1997). that after 30 min imbibition, the aromatic BA was the only
Seedling establishment follows radicle emergence. In the cytokinin found in significant concentrations while mT, iP,
present study, the samples harvested on day 0 after 30 min iPR, cZR and cZRMP were detected at very low levels.
and 5 h imbibition are two ‘‘snapshots’’of cytokinin levels After 5 h imbibition, cZRMP had greatly increased in
during germination. By day 1, germination of the pea seeds concentration while there were only small increases in the
was complete as seen by radicle emergence and the sub- other iP and cZ derivatives.
sequent samples show cytokinin levels in the root during The biosynthetic rates (t/t ratio) of the cZ-type cytoki-
seedling establishment. nins remained low for the duration of the experiment as
Wang and Sponsel (1985 in Emery and Atkins 2006) determined by in vivo deuterium labelling followed by
noted that despite seeds being a rich source of cytokinins, LC(+)-MS/MS approach (Table 2). These rates did not
no cytokinins had been identified in seeds of any pea correspond to the high endogenous levels of the cZ-type
species using a variety of techniques including bioassays, cytokinins found in the pea radicle and seedling roots. It is
immunoassays and mass spectrometry. Recent advances in possible that these cZ-forms are released from an as yet
the analytical techniques used in plant growth regulator unidentified conjugate that is present in the dry seed or
detection are very sensitive and allow for the identification originate from tRNA degradation. This indirect pathway of
of cZ-isomers from their trans forms. cis-Z forms are now cytokinin production by the isomerization of cZ to the
routinely found in plant tissues and in some cases, are the highly active tZ cannot be ignored especially in tissues

123
1286 Planta (2008) 227:1279–1289

Fig. 5 Changes in
400 a) t ZRMP b) c ZRMP
concentration of cytokinin
ribotides; a trans-zeatin 350 Root tip
riboside-50 -monophosphate 300 Root free zone
(tZRMP, a), cis-zeatin riboside- Root zone

CYTOKININ CONCEN TRATION (pmol g-1 DW)

250
50 -monophosphate (cZRMP, b), Secondary roots
dihydrozeatin riboside-50 - 200 Radicle
monophosphate (DHZMP, c) 150
and N6-(2-
isopentenyl)adenosine-50 - 100 Radicle
monophosphate (iPRMP, d) in

< LO D

<LO D
50

< LO D
<L O D

< LO D
pea seedlings measured over
0
9 days of incubation. No
aromatic cytokinin ribotides 400 c) DHZRMP d) iPRMP
were detected. LOD limit of
350
detection, error bars represent
SD (n = 3) 300

250

200

150

100
Radicle

<L O D
< LOD
< LO D

< LO D
<LOD

<LOD

<LOD

<LOD
<L O D
50 Radicle

0
1 3 6 9 1 3 6 9

in
in

h
h

m
m

5
5

TIME (days) TIME (days)

30
30

with high cZ concentrations (Sakakibara and Takei 2002; There is still speculation as to the physiological role of
Sakakibara 2006) such as the pea seeds used in this study. cZ derivatives. In bioassays, cZ generally has lower or no
Recent studies with Arabidopsis suggest that the mevalo- activity compared with tZ and iP (Sakakibara 2006). cis-Z
nate pathway may also be involved in the biosynthesis of conjugates were originally believed to be relatively inac-
cZ derivatives (Kasahara et al. 2004). cis-Z levels had an tive storage forms that were readily converted to the more
inverse correlation to iP and tZ levels, indicating that cZ- biologically active trans-forms (Emery et al. 1998). A cis–
type cytokinins are produced by an alternative pathway trans isomerase enzyme has been partially purified from
with the first step catalysed by tRNA-isopentenyltransfe- immature Phaseolus seeds to support this theory (Bassil
rases (IPT; AtIPT2 and AtIPT9; Miyawaki et al. 2004, et al. 1993). Two cZOG-specific genes have been isolated
2006). Although these enzymes are expressed ubiquitously, from maize kernels indicating that the cis-isomers could
they show stronger expression in proliferating tissue such have a metabolic pathway served by cis-specific enzymes
as root meristems (Miyawaki et al. 2004). This expression parallel to the trans-isomer pathway (Martin et al. 2001;
would allow the independent regulation of cZ derivative Veach et al. 2003). Yonekura-Sakakibara et al. (2004)
levels from tZ levels and supports the idea that cZ cyto- showed that the histidine kinases found in maize responded
kinins are derived mainly from modified tRNA (Kasahara to cZ, suggesting that cZ plays a role in cytokinin signal-
et al. 2004; Sakakibara 2006). ling in maize. The physiological significance of cis isomers

Table 2 Biosynthetic rates of the cytokinin ribotides isopentenylad- (cZRMP) and the cytokinin ribosides isopentenyladenosine (iPR),
enosine-50 -monophosphate (iPRMP), trans-zeatin riboside-50 - trans-zeatin riboside (tZR) and cis-zeatin riboside (cZR) in develop-
monophosphate (tZRMP) and cis-zeatin riboside-50 -monophosphate ing pea radicles and seedling roots
Treatment Biosynthetic rate (t/t ratio)
iPRMP iPR tZRMP tZR cZRMP cZR

Seeds (24 h) 0.03 ± 0.04 0.03 ± 0.06 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.00 ± 0.00
Seeds (48 h) 0.03 ± 0.03 0.05 ± 0.04 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.00 ± 0.00
Roots (day 6) 1.01 ± 0.14 0.83 ± 0.06 0.55 ± 0.02 0.36 ± 0.05 0.07 ± 0.01 0.04 ± 0.00
Roots (day 9) 1.03 ± 0.16 2.47 ± 0.53 1.04 ± 0.24 1.47 ± 0.54 0.05 ± 0.08 0.05 ± 0.10

Results are shown as mean ± SD (n = 4)

123
Planta (2008) 227:1279–1289
in pea radicles and roots needs to be further investigated.
Their rapid appearance, especially cZRMP after 5 h
imbibition, suggests that they may possibly have an
important regulatory function in radicle elongation and
early seedling establishment.
Aromatic cytokinins have been identified in a number of
plant tissues (see Taylor et al. 2003) although as yet,
nothing is known about their biosynthesis and regulation
(Sakakibara 2006). Although isoprenoid and aromatic
cytokinins have an overlapping spectrum of biological
activity, they are not considered as alternative forms of the
same signals (Strnad 1997). They are believed to be
involved in the metabolism and development of mature
tissues rather than in the stimulation of cell division
(Kamı́nek et al. 1987) and play a role in retarding senes-
cence (Strnad 1997).
In the present study, BA was detected in high con-
centrations in the radicles of pea seeds imbibed for
30 min and increased in concentration after 5 h imbibi-
tion. With seedling establishment, BA slowly decreased
over time with no other BA derivatives being detected
(Fig. 1e). Further, BA remained localized in the root tip
and root free zone with much lower concentrations in the
root zone and was not detected in the secondary roots.
Similar results were obtained in Tagetes minuta achenes
where high BA concentrations were recorded in the dry
achenes and decreased rapidly upon imbibition with no
indication of interconversion (Stirk et al. 2005). The
results suggest that BA was either actively used or
transported away from the root tip to the aerial organs, as
there was no indication of interconversion to other BA
conjugates. P. sativum is able to transport BA from the
roots to the shoots (Procházka and Jacobs 1984). The
biosynthetic rate of BA in the pea seeds could not be
measured, as there is not yet a method available. Unlike
isoprenoid cytokinins where the side chain is synthsized
de novo, the benzyl ring of aromatic cytokinins appears to
be recycled and thus there is almost zero incorporation of
deuterium into the aromatic side chain and purine ring. To
date, there is not a sensitive enough method to measure
such low levels of incorporation.
The changing concentrations of BA in the pea seeds are
consistent with the presence of cytokinin binding proteins
(CBPs), the first of which was described from wheat grains
as CPB-1 (Fox and Erion 1975). CBP-1 accumulated rap-
idly in wheat embryos and accounted for 10% of the
soluble embryo protein content at maturation (Brinegar
et al. 1988). Cytokinin binding proteins are closely asso-
ciated with seed storage proteins of a vicilin class, have the
highest affinity for aromatic cytokinins, especially BA, and
are localized in the protein bodies of the tissues sur-
rounding the embryonic axis (Brinegar and Fox 1985). As
they have a relatively low binding of isoprenoid cytokinins
1287
compared to aromatic cytokinins (Keim et al. 1981), it has
been hypothesized that CBPs may function either in sig-
nalling initiation of germination or in temporal
immobilization of aromatic cytokinins during grain
development, thus preventing premature embryo cell
division (Kamı́nek et al. 2003). In many species, radicle
protrusion is due to cell elongation with cell division only
commencing a few hours after radicle protrusion (Cantliffe
et al. 1984). The high concentrations of BA in the pea
radicles in the present study suggest that perhaps BA plays
a similar role in preventing premature cell division. A
similar pattern was also found in Tagetes minuta achenes
(Stirk et al. 2005). This pattern may perhaps be a wide-
spread phenomenon in seeds that has been overlooked in
the past and requires further investigation. The presence of
BA in pea radicles was therefore unequivocally verified by
comparison of the Q-Tof MS (Fig. 2) data and chromato-
graphic retention times with those of authentic standards.
From the results mentioned earlier, one may consider the
possibility that some embryo CBPs sequester cytokinins
within protein bodies to control their effective levels during
embryogenesis or to store them for use in the initiation of
germination. Seed storage tissues store proteins, fatty acids,
starch and vitamins (Hagan and Higgins 2006) and they
could possibly do the same with cytokinins.
Other isoprenoid cytokinins (iP-, tZ- and DHZ-types)
were first detected in significant quantities in the pea
seedlings after radicle emergence from day 1 onwards
(Figs. 1, 2, 3, 4, 5). Several other pathways for the de novo
biosynthesis of isoprenoid cytokinins have been elucidated,
being produced through the methylerythritol pathway
(Kasahara et al. 2004). The initial step in isopentenylade-
nosine-dependent pathways is the N-prenylation of AMP,
ADP or ATP with dimethylallyl pyrophosphate (DMAPP)
or hydroxymethylbutenyl diphosphate (HMBDP) to
form isopentenyladenosine-50 -triphosphate (iPRTP), iso-
pentenyladenosine-50 -diphosphate (iPRDP) or iPRMP,
respectively (Kakimoto 2001; Takei et al. 2001; Sakakibara
2006). These reactions are catalysed by tissue- and sub-
strate-specific adenosine phosphate-IPT. This reaction is
followed by the hydroxylation of the side chain to form tZ
conjugates (Sakakibara 2006). Åstot et al. (2000b) proposed
an isopentenyladenosine-independent pathway with tZRMP
being the first cytokinin metabolite formed by IPT using an
unidentified hydroxylated side-chain precursor derived
from the mevalonate pathway. Sakakibara (2006) suggested
that tZ biosynthesis via this pathway may be mediated by
cis–trans isomerization of cZ derivatives. The various
biosynthetic pathways are likely to operate together in a
plant but may vary in a tissue- and time-dependent manner
in response to environmental stimuli (Åstot et al. 2000b;
Takei et al. 2001). For example, ZRMP levels were not
altered in mutant Arabidopsis with impaired lateral root
123
1288
formation suggesting that the major site for ZRMP syn-
thesis is in the aerial parts of the plant. However, reduced
iPRMP levels suggested that the isopentenyladenosine-
dependent pathway dominated in the root meristems
(Nordström et al. 2004a).
High concentrations of both iPRMP and tZRMP were
measured in the pea seedling roots. While iP-types were
detected in both the root tip and the elongating secondary
roots, no tZ-types were detected in the root tip. The lower
concentrations of the DHZ-type cytokinins mirrored the
distribution of their corresponding tZ forms, suggesting
that there could be conversion between tZ and DHZ forms.
The biosynthetic rates of both ribotides and ribosides of iP
and tZ were low during germination but increased on day 6
(Table 2). This increase corresponded to secondary root
elongation, suggesting that the secondary roots could be a
major site of cytokinin biosynthesis in pea roots. There is
an increase in expression of the isopentenyltransferase
genes in the root cap of both developing primary roots and
secondary roots of Arabidopsis suggesting the possible role
of the root cap in cytokinin production (Miyawaki et al.
2004). Recently, Kurakawa et al. (2007) identified an LOG
gene in rice shoot meristems that encodes an enzyme which
is able to directly convert relatively inactive cytokinin ri-
bosides to their corresponding free-base forms by specific
phosphoribohydrolase activity. If the same gene is found in
the root meristems of the pea plants used in this study, the
high concentrations of the ribotides, especially iPRMP in
the root tip (Fig. 5d) could provide a source for the rapid
conversion to the more biologically active free bases.
The gradient of cytokinins in pea roots has been well
studied with highest levels detected in the root tip. Decapi-
tation of the root tip disrupts the spatial organization of the
root (Short and Torrey 1972; Stirk and van Staden 2001). The
distance of the secondary roots from the root tip is regulated
by cytokinin:auxin ratios. High cytokinins in the root tip are
antagonistic to IAA and thus inhibit secondary root initia-
tion. Secondary root initiation only occurs above the
elongation zone where cytokinin concentrations are lower
(Aloni et al. 2006). In the current study, such a gradient was
observed in the pea roots with iP derivatives where their
highest concentrations were detected in the root tip and the
lowest concentrations in the root zone. The presence of high
concentrations of tZR in the root-free zone and root zone
(Fig. 3a) may be due to the translocation of tZR to the aerial
shoots. Zeatin riboside is found in the xylem sap of P. sati-
vum (Beveridge et al. 1997). The secondary roots also had
high concentrations of tZ, iP and iP conjugates. Perhaps the
high levels of these cytokinins have a similar function in
preventing further branching of the secondary roots.
In conclusion, the levels and types of cytokinins fluc-
tuated considerably in the various components of the pea
seedling roots over time. The only cytokinins detected
123
Planta (2008) 227:1279–1289
during germination were cZ conjugates and the aromatic
BA. The biosynthetic rates of cZ derivatives remained low
throughout the experiment. As there is a high rate of RNA
and protein synthesis during germination, these cytokinins
may possibly be due to degradation processes. Other iso-
prenoid cytokinins (iP, tZ and DHZ) were only detected
after radicle emergence with the highest biosynthetic rates
being measured from day 6 onwards to correspond with
secondary root emergence. While iP derivatives were
measured in both the root tip and the secondary roots, tZ
and DHZ derivatives were only measured in the secondary
roots. These results highlight the great complexity of the
various cytokinin forms in roots and their possible multiple
roles in regulation root development.
Acknowledgments Georgina Arthur, Marnie Light and Nicky
Taylor, University of KwaZulu-Natal are thanked for their assistance
in harvesting the seedlings and Hana Martinková and Petra
Amarkorová, Palacký Univeristy and Institute of Experimental Bot-
any AS CR for their help with cytokinin analyses. The National
Research Foundation, South Africa and the Czech Ministry of Edu-
cation (grant No. MSM 6198959216) are thanked for financial
assistance.
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