Annals of Botany 112: 41–55, 2013
doi:10.1093/aob/mct092, available online at
High cytokinin levels induce a hypersensitive-like response in tobacco
Jan Nova´k1,2, Jaroslav Pavlu˚1,2, Ondrˇej Nova´k3, Vladimı´ra Nozˇkova´-Hlava´cˇkova´4, Martina Sˇpundova´4,
ˇ erny´1,2 and Brˇetislav Brzobohaty´1,2,*
Jan Hlavinka4, Sˇa´rka Koukalova´1,2, Jan Skala´k1,2, Martin C
Department of Molecular Biology and Radiobiology, Mendel University in Brno, Zemeˇdeˇlska´ 1, CZ-613 00 Brno, Czech Republic,
CEITEC – Central European Institute of Technology, Mendel University in Brno, Zemeˇdeˇlska´ 1, CZ-613 00 Brno, Czech Republic,
Laboratory of Growth Regulators, Palacky´ University and Institute of Experimental Botany AS CR, Sˇlechtitelu˚ 11, CZ-783 71, Czech
Republic and 4Centre of the Region Hana´ for Biotechnological and Agricultural Research, Department of Biophysics, Palacky´
University Olomouc, Sˇlechtitelu˚ 11, CZ-783 71 Olomouc, Czech Republic
* For correspondence. E-mail [email protected]
Received: 8 January 2013 Revision requested: 7 February 2013 Accepted: 11 March 2013 Published electronically: 3 May 2013
Key words: Cytokinin, hypersensitive response, hydrogen peroxide, lipid peroxidation, pathogenesis-related
proteins, salicylic acid, jasmonic acid, abscisic acid, photosynthesis, stomatal conductance, non-photochemical
quenching, Nicotiana tabacum.
Cytokinins were first identified by their ability to promote cell
division in tobacco tissue cultures (Miller et al., 1955). They
have since been reported to promote shoot development, delay
leaf senescence, contribute to stress and pathogen responses,
and serve as important signals for co-ordinating growth rates
throughout the plant (Argueso et al., 2009; Werner and
Schmu¨lling, 2009; Ha et al., 2012). The modulation of cytokinin
metabolism or signalling could thus potentially be a powerful
tool for improving crop yield. For example, rice plants that
have elevated cytokinin levels due to a decrease in the expression
of the cytokinin-degrading enzyme cytokinin oxidase/dehydrogenase can produce more grains per plant because of changes
in their inflorescence architecture (Ashikari et al., 2005).
Increasing leaf longevity via senescence-specific overexpression
of a key cytokinin biosynthetic gene (ipt) was shown to increase
biomass and seed production (Gan and Amasino, 1995), prolong
the post-harvest shelf life of leaf crops (McCabe et al., 2001) and
induce extreme drought tolerance (Rivero et al., 2007).
Moreover, cytokinins play critical roles in plant – microbe
interactions. In nitrogen-fixing plants, cytokinin receptors
are important for symbiotic nitrogen fixation. Specifically,
cytokinin receptor loss-of-function mutants are unable to form
nodules (Gonzalez-Rizzo et al., 2006; Murray et al., 2007),
while gain-of-function mutants form nodules spontaneously
(Tirichine et al., 2007). Several lines of evidence indicate that
cytokinins have a wide range of functions in plant – pathogen
interactions. Pathogen infection has been reported to cause
changes in cytokinin levels. For example, some pathogens
seem to use their ability to produce cytokinins to facilitate
disease development. Agrobacterium tumefaciens is the causal
# The Author 2013. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
Downloaded from by guest on March 30, 2015
† Background and Aims Cytokinins are positive regulators of shoot development. However, it has previously been
demonstrated that efficient activation of the cytokinin biosynthesis gene ipt can cause necrotic lesions and wilting
in tobacco leaves. Some plant pathogens reportedly use their ability to produce cytokinins in disease development.
In response to pathogen attacks, plants can trigger a hypersensitive response that rapidly kills cells near the infection
site, depriving the pathogen of nutrients and preventing its spread. In this study, a diverse set of processes that link ipt
activation to necrotic lesion formation were investigated in order to evaluate the potential of cytokinins as signals and/
or mediators in plant defence against pathogens.
† Methods The binary pOp-ipt/LhGR system for dexamethasone-inducible ipt expression was used to increase
endogenous cytokinin levels in transgenic tobacco. Changes in the levels of cytokinins and the stress hormones
salicylic, jasmonic and abscisic acid following ipt activation were determined by ultra-performance liquid
chromatography –electrospray tandem mass spectrometry (UPLC-MS/MS). Trends in hydrogen peroxide content
and lipid peroxidation were monitored using the potassium iodide and malondialdehyde assays. The subcellular distribution of hydrogen peroxide was investigated using 3,3′ -diaminobenzidine staining. The dynamics of transcripts
related to photosynthesis and pathogen response were analysed by reverse transcription followed by quantitative
PCR. The effects of cytokinins on photosynthesis were deciphered by analysing changes in chlorophyll fluorescence
and leaf gas exchange.
† Key Results Plants can produce sufficiently high levels of cytokinins to trigger fast cell death without any intervening chlorosis – a hallmark of the hypersensitive response. The results suggest that chloroplastic hydrogen peroxide
orchestrates the molecular responses underpinning the hypersensitive-like response, including the inhibition of
photosynthesis, elevated levels of stress hormones, oxidative membrane damage and stomatal closure.
† Conclusions Necrotic lesion formation triggered by ipt activation closely resembles the hypersensitive response.
Cytokinins may thus act as signals and/or mediators in plant defence against pathogen attack.
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
Plant material and treatments
Transgenic plants CaMV35S . GR . ipt ( pOp6-ipt/LhGR-N,
lines 303 and 307; Sˇa´malova´ et al., 2005) and the corresponding
wild-type Nicotiana tabacum ‘SR1 Petit Havana’ were grown in
an AR-36L growth chamber (Percival Scientific) with 16 h light
at 24 8C/8 h dark at 21 8C under a photosynthetic photon flux
density of 100 mmol photons m22 s21 provided by cool white
fluorescent lamps (Philips). All experiments were performed
using 5-week-old plants. A dexamethasone (DEX) (Sigma) solution with a final concentration of 20 mM was prepared by diluting
a 20 mM stock solution in 96 % ethanol (Lachema, Czech
Republic) with tap water. A 50 mL aliquot of the diluted solution
was applied to the soil by watering 3 h before the start of the dark
period. Control plants were watered with 0.096 % ethanol in tap
water (mock). Samples were collected in the middle of the light
period from non-damaged tissue at the indicated times after DEX
application, frozen in liquid nitrogen and stored at – 80 8C. For
trans-zeatin (t-Z) feeding, leaves were detached from wild-type
plants, immersed via petioles into t-Z solutions in tap water of
concentrations ranging from 10 mM to 2.5 mM and incubated
for 3 d under the light and temperature regime outlined above.
Controls were fed with 1 % dimethylsulfoxide (DMSO; v/v) in
tap water. To minimize gas exchange, lanolin was applied to
the leaf surfaces according to Mateo et al. (2004).
Quantification of phytohormones
For endogenous cytokinin analysis, plant material with a fresh
weight of approx. 0.2 g was extracted and purified according to
the method described by Nova´k et al. (2003). A cocktail of isoprenoid deuterium and 13C6-labelled standards (Olchemim)
was added (each at 1 pmol per sample) to check the recovery
during purification and to validate the determination. The cocktail consisted of [13C5]c-Z, [13C5]t-Z, [2H5]t-ZR, [2H5]t-Z7G,
[2H5]t-Z9G, [2H5]t-ZOG, [2H5]t-ZROG, [2H5]t-ZMP, [2H3]
DHZ, [2H3]DHZR, [2H3]DHZ9G, [2H7]DHZOG, [2H3]DHZMP,
[2H6]iP, [2H6]iPR, [2H6]iP7G, [2H6]iP9G and [2H6]iPMP (for a
list of abbreviations, see Supplementary Data Table S2). The
samples were purified using two ion-exchange chromatography
steps (SCX followed by a DEAE-Sephadex column in conjunction with SPE C18-cartridges) and immunoaffinity purification.
The cytokinin levels in each sample were quantified by ultraperformance liquid chromatography – electrospray tandem
mass spectrometry (UPLC-MS/MS) (Nova´k et al., 2008). Endogenous salicylic acid (SA), jasmonic acid (JA) and abscisic acid
(ABA) were determined in samples with fresh weights of 0.3 g
using a combination of an octadecylsilica column (500 mg;
Agilent Technologies) and a DEAE-Sephadex (Sigma
Aldrich) column with a Sep-Pak C18 cartridge (360 mg;
Waters). Plant material was extracted overnight in
80 % methanol, and 100 pmol of [2H4]SA (Cambridge Isotope
Laboratories), 50 pmol of [2H6]ABA and [2H6]JA (Olchemim)
were added as internal standards. Quantification was performed
as described by Bergougnoux et al. (2009). Separation was performed on an Acquity UPLC System (Waters) equipped with a
Luna phenyl–hexyl column (250 × 2.0 mm, 5 mm; Phenomenex,
Torrance, CA, USA), and the effluent was introduced into the
Downloaded from by guest on March 30, 2015
agent of crown gall disease in a number of dicot species, including some important crops. The symptoms of the disease are
caused by the insertion of T-DNA from a bacterial Ti plasmid
into the plant genome. This T-DNA carries genes necessary
for the biosynthesis of cytokinins (ipt), auxin and opines.
While high cytokinin and auxin levels convert the transformed
cells into rapidly growing tumours, opines secreted from
the transformed cells serve as a source of carbon and nitrogen
for the Agrobacterium (Chilton et al., 1977). Pathogen(Plasmodiophora brassicae) derived cytokinins are also
involved in the development of clubroot disease (Devos et al.,
2006; Siemens et al., 2006), and transgenic plants that overexpress cytokinin oxidase/dehydrogenase show less severe
disease symptoms (Siemens et al., 2006). On the other hand,
beans inoculated with the viral pathogen White clover mosaic
potexvirus exhibited reduced cytokinin levels (Clarke et al.,
1999), and treatment of the inoculated bean plants with exogenous cytokinin resulted in reduced replication of the White clover
mosaic potexvirus concomitant with upregulation of defence response genes such as those encoding pathogenesis-related (PR)
proteins (Clarke et al., 1998).
Measures adopted by plants against disease-causing microorganisms include constitutive defences (in the form of antimicrobial secondary metabolites) and induced defence
responses that require detection of the pathogen by the plant,
which is followed by the rapid activation of defence-related
genes. A plant – pathogen interaction that triggers the full
defence syndrome is said to be incompatible. A common
defence is the hypersensitive response, in which cells immediately surrounding the infection site die rapidly and thus deprive the
pathogen of nutrients, preventing its spread. The hypersensitive
response is preceded by the rapid accumulation of reactive
oxygen species (ROS) (Dangl et al., 1996; Love et al., 2008;
O’Brien et al., 2012). A characteristic feature of defence
responses is the expression of PR proteins (Rushton and
Somssich, 1998), which are a diverse group of enzymes, antifungal agents and secondary signalling components.
Despite their well documented function in delaying senescence, there are several lines of evidence linking cytokinins to
the induction of cell death. It was shown that Arabidopsis and
carrot cell cultures cultivated in a medium with high cytokinin
content exhibit reduced growth and show hallmarks of programmed cell death (Carimi et al., 2003). The authors also
showed that cytokinins promote premature senescence and the
onset of apoptosis hallmarks in Arabidopsis plantlets cultivated
in vitro. Similar results were obtained using tobacco BY-2 cells
treated with the cytokinins isopentenyladenine and benzyladenine, as well as the corresponding ribosides (Mlejnek et al.,
2002, 2003, 2005). Necrosis in old leaves was reported in transgenic tobacco and maize, in which targeted cytokinin accumulation in these leaves was achieved by placing ipt expression under
the control of senescence-specific promoters (Robson et al.,
2004; Wingler et al., 2005).
We have previously reported necrotic lesion formation in the
leaves of transgenic tobacco plants within 3 d of ipt activation
(Sˇa´malova´ et al., 2005). Here, we employed this system to investigate in detail the diverse processes that lead from ipt activation
to necrotic lesion formation. Based on the data obtained, we
discuss the potential roles of cytokinins in the hypersensitive response to pathogen attacks.
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
electrospray ion source of a XevoTM TQ MS (Waters) triple
quadrupole mass spectrometer (Waters).
Determination of leaf dry matter content and chlorophyll
Leaf dry matter content (LDMC) was measured by drying
1 – 2 g of fresh plant material for 5 d in a drying oven
(Memmert) at 70 8C and then for 2 d at 105 8C when constant
weight of samples was reached.
Samples for chlorophyll determination were cut with a cork
borer and analysed according to Wellburn (1994). Specifically,
the samples were transferred into tubes containing 2 mL of
99.5 % DMSO (Sigma-Aldrich) and incubated for 2 h at 50 8C
with occasional shaking. Their absorbance was then read at
649 and 665 nm (Helios b, Chromspec) and these measurements
were used to calculate the sample’s chlorophyll content
(Wellburn, 1994).
Hydrogen peroxide was quantified using a slightly modified
variant of the method reported by Alexieva et al. (2001).
Leaves (300 mg f. wt) were homogenized to powder in liquid nitrogen, extracted in 3 mL of 0.1 % trichloroacetic acid (TCA) and
centrifuged at 10 000 g at 4 8C for 10 min. The supernatant was
separated and mixed with 0.1 M potassium phosphate buffer
( pH 7) and 0.5 M potassium iodide (Fluka) solution at a ratio of
1:1:2 (v/v/v). The resulting mixture was allowed to react for
30 min in the dark, after which its absorbance at 390 nm was
measured. The hydrogen peroxide content of the sample was
then calculated using a standard curve.
The subcellular distribution of hydrogen peroxide was visualized by endogenous peroxidase-dependent histochemical staining using 3,3′ -diaminobenzidine (DAB) (Sigma) according to
Poga´ny et al. (2004) with slight modifications. Leaves were incubated in 2.5 mM Tris-phosphate buffer ( pH 7.8) supplemented
with 2.5 mM DAB for 2 h. The incubated leaves were then decolorized by incubation in a clearing solution comprising 79.85 %
ethanol, 20 % chloroform (Penta, Czech Republic) and 0.15 %
TCA (Sigma). The presence of hydrogen peroxide is indicated
by the formation of a brown precipitate.
The extent of lipid peroxidation within samples was determined by estimating their malondialdehyde (MDA) content
according to a modification of the method developed by Heath
and Packer (1968). Samples were extracted with 0.1 % TCA
using the procedure that was employed when determining hydrogen peroxide levels. A reaction mixture comprising 0.5 mL of
supernatant, 0.5 mL of a 0.1 M potassium phosphate buffer
( pH 7.0) and 1 mL of 20 % TCA containing 0.5 % thiobarbituric
acid (Sigma) was then prepared and heated on a water bath at
95 8C for 30 min, after which it was rapidly cooled in an ice
bath. The cooled mixture was centrifuged at 10 000 g at 4 8C
for 5 min and the absorbance of supernatant at 532 and 600 nm
was determined. The non-specific absorbance at 600 nm was
subtracted from the absorbance at 532 nm, and the concentration
of MDA was calculated based on an extinction coefficient of
155 mM21 cm21.
RT –qPCR analysis
Steady-state transcript levels of genes coding for bacterial
isopentenyl transferase (ipt), chlorophyll a/b-binding protein
(CAB), ferredoxin:NADP oxidoreductase (FNR1), violaxanthin
de-epoxidase (VDE), pathogenesis-related protein 1b (PR-1b)
and acidic phosphatase (PR-Q) were quantified by reverse transcription followed by quantitative PCR (RT – qPCR) using the
fluorescent dye SYBR Green I. Leaves of 5-week-old tobacco
were frozen in liquid nitrogen and total RNA was extracted
using the TRIzol reagent (Invitrogen). The resulting total RNA
extract was treated with TurboDNase (Ambion) to remove residual DNA. Reverse transcription was performed using the
SuperScriptII reverse transcriptase (Invitrogen) and an
oligo(dT) primer. qPCR was performed using the Rotor-Gene
6000 real-time analyzer (Corbett Research, Australia) with the
primers and conditions listed in Supplementary Data Table S1.
The amplification of a single specific product was verified by
melting curve analysis and agarose gel electrophoresis. The
measured expression levels were normalized by geometric averaging using the GeNorm VBA applet for Microsoft Excel
(Vandensompele et al., 2002), with actin (Tob66) and EF-1a
as the reference genes.
Leaf gas exchange
Leaf gas exchange was measured using an LI-6400 open
gas-exchange system (LI-COR, Lincoln, NE, USA) on the
third or fourth attached leaf (counted from the plant base) after
30 min of dark adaptation. Light induction of photosynthesis
and transpiration was measured over 45 min of exposure to
actinic light (330 mmol photons m22 s21) that was switched
on after a 5 min period for the leaves to acclimate to the
chamber conditions. The chamber conditions were as follows:
mass flow of air 200 mmol s21, relative air humidity 50 %,
block temperature 24.5 8C, CO2 concentration 390 mmol mol21.
The CO2 response of photosynthesis was measured under an air
mass flow of 300 mmol s21 at nine different ambient CO2 concentrations (Ca); the starting Ca was 400 mmol mol21, followed by
300, 200, 100, 50, 400, 600, 800, 1000 and 1200 mmol mol21.
The intercellular CO2 concentration (Ci) was calculated using the
function described by von Caemmerer and Farquhar (1981). The
carboxylation efficiency of Rubisco was indicated by the slope of
the initial linear part of the A/Ci curve.
Chlorophyll fluorescence parameters
Slow induction kinetic parameters were measured on the
adaxial sides of the third or fourth attached leaves using a
& P700
Photosynthesis Analyzer (Heinz Walz GmbH, Effeltrich,
Germany), which can be used to monitor chlorophyll fluorescence and changes in P700 absorbance simultaneously. Plants
were pre-darkened for 30 min and then exposed to measuring
light (approx. 1 mmol photons m22 s21) and red actinic light
(approx. 100 mmol photons m22 s21) for 10 min together with a
series of 0.5 s saturation pulses (6000 mmol photons m22 s21,
red light) every 45 s. The non-photochemical chlorophyll fluorescence quenching (NPQ), the relative rates of electron transport
via photosystem I (PSI) and PSII – ETR (I) and ETR (II), respectively – and the quantum yield of non-photochemical energy dissipation due to donor side limitations of PSI [Y(ND)] at
steady-state were estimated (Klughammer and Schreiber,
2008). Images of steady-state NPQ were recorded using a
Downloaded from by guest on March 30, 2015
Determination of hydrogen peroxide and lipid peroxidation
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
Statistical analysis
Three biological replicates were used in each experiment. In
each biological replicate, three random samples were collected
from each of three plants. The resulting nine samples were analysed in a single technical replicate in all experiments except
those involving RT – qPCR, in which case two technical replicates were used. Student’s t-test was used to evaluate the statistical significance of the results obtained. The standard deviation
of the measured ratios was calculated using the equation:
SD1 2
SD2 2
SD =
ipt activation results in a hypersensitive-like response in the leaves
of tobacco plants
The binary pOp-ipt/LhGR system for DEX-inducible ipt expression (Sˇa´malova´ et al., 2005) was used to increase endogenous
cytokinin levels in transgenic tobacco plants, and the resulting
morphological, histological, biochemical and molecular
changes were investigated. Two independent CaMV35S .
GR . ipt lines (303 and 307) were cultivated for 5 weeks under
standard long-day photoperiod conditions and exhibited no detectable phenotypic differences relative to wild-type plants
during this period. However, when the transgenic plants were
watered once with 20 mM DEX in tap water, slightly translucent
Dynamics of the cytokinin pool upon ipt activation in the tobacco
To investigate the dynamics of the cytokinin pool following
ipt activation, the cytokinin bases and their metabolites were analysed in samples of expanded horizontal leaves that did not
3 cm
F I G . 1. Phenotype of CaMV35S . GR . ipt plants after treatment with dexamethasone. Five-week-old CaMV35S . GR . ipt lines 303 (A, C), 307 (B) and the wild
type (D) after 4 d of treatment with 50 mL of 20 mM dexamethasone (A, B, D) or 0.096 % ethanol (C).
Downloaded from by guest on March 30, 2015
zones appeared on expanded horizontal leaves around 50 h after
exposure to DEX. The translucent zones developed into lesions
over the following 10 h (Fig. 1). No marks of chlorosis were apparent on these leaves and no statistically significant decrease in
chlorophyll content occurred (Supplementary Data Fig. S1).
Massive lesion formation continued until the fourth day of DEX
application and then ceased. Lesion formation was associated
with wilting, and both these processes resulted in the complete
collapse of the expanded horizontal leaves in plants of line 303,
which gradually died over the following 3 weeks. The extent of
lesion formation and wilting was less severe in line 307 (Fig. 1),
and plants of this line recovered from the effects of the single dose
of 20 mM DEX, developed normally afterwards and even produced viable seeds. Lesion formation was dose dependent. In
the more responsive line 303, a single treatment with 1 mM
DEX was sufficient to provoke some lesion formation, but
lower DEX concentrations were non-effective (Fig. 2).
Young, recently opened upward-facing leaves underwent
some distinctive processes immediately after exposure to
DEX. The leaves initially curled and then (on the fourth day
after being treated with DEX) developed chlorosis (Fig. 3).
These leaves did not develop substantial necrotic lesions.
Interestingly, a slight but significant transient increase in chlorophyll content was seen in line 307 following activation.
Root growth was also negatively affected in the transgenic
plants exposed to DEX for 4 d. The fresh weight of the root
system harvested from DEX-treated line 303 was lower by
25 % (P , 0.05) compared with the mock-treated line 303,
while DEX treatment did not result in any statistically significant
difference in the root system of wild-type plants.
FluorCam 700MF imaging system (Photon Systems Instruments,
Czech Republic). The adaxial side of the third or fourth attached
leaf was irradiated for 10 min with actinic light (100 mmol
photons m22 s21) and the measurement protocol described by
Prokopova´ et al. (2010) was applied.
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
3 cm
10 mM
20 mM
1 mM
0·5 mM
5 mM
0·25 mM
2 mM
0·1 mM
Chlorophyll content
(ratio +DEX/–DEX, %)
** **
1 cm
Line 303
Line 307
wt (SR1)
F I G . 3. Time course of chlorophyll content in young leaves of CaMV35S .
GR . ipt plants following ipt activation. Five-week-old CaMV35S . GR .
ipt lines 303, 307 and the wild type (SR1) were induced with 50 mL of 20 mM
dexamethasone. Samples for chlorophyll analysis were collected at 19 h (1
DAT), 43 h (2DAT), 67 h (3 DAT) and 91 h (4 DAT) after treatment with dexamethasone. (A) The chlorophyll content of plants treated with dexamethasone
(+DEX) is expressed as a percentage of that observed in plants treated with
0.096 % ethanol (– DEX) and all reported values represent the means of nine measurements. Error bars indicate the s.d. One and two asterisks indicate statistically
significant differences between +DEX and –DEX plants at P ≤ 0.05 and P ≤
0.01, respectively, based on the t-test. (B) Typical leaves of DEX-treated plants.
contain lesion areas (Fig. 4) by UPLC-MS/MS. Dramatic
increases in the levels of both cytokinin bases and their ribosides
and ribotides were observed within 19 h of exposure to a single
dose of 20 mM DEX (first day after DEX treatment; 1 DAT).
These increases levelled off over the following 24 h (2 DAT),
reaching values about four orders of magnitude higher than
those observed in non-activated samples. In absolute terms, the
levels of cytokinin free bases in the activated leaves reached
30 nmol g21 f. wt, while the levels of the ribosides and ribotides
rose to 64 nmol g21 f. wt. O-Glucosylation represented a significant part of conjugative inactivation of free cytokinins.
While there was a very significant increase in the levels
of cytokinin-O-glucosides and cytokinin-N9-glucosides by
1 DAT, it remained much less pronounced than the increase in
the levels of the corresponding free bases until 2 DAT. From
3 DAT onwards, the levels of cytokinin-O-glucosides were of
the same order of magnitude as those of free bases, but those of
the cytokinin-N9-glucosides were around three orders of magnitude lower. The cytokinin pool was dominated by t-Z and its
metabolites (Supplementary Data Table S2). In general, following ipt activation, the cytokinin pool was consistently somewhat
higher in line 303 than in line 307, which is consistent with the
stronger phenotypic response observed in line 303: as discussed
above, a DEX solution of only 1 mM was sufficient to induce
lesion formation in line 303. Analysis of the cytokinin pool
(Supplementary Data Fig. S2) revealed that in line 303, on the
third day after a single application of 1 mM DEX, the levels of
free cytokinin bases, their riboside and ribotides, and O- and
N9-glucosides were only 2-, 9-, 10- and 3-fold lower, respectively, than those observed following treatment with 20 mM DEX.
This implies that the levels of free biologically active cytokinins,
i.e. free bases, must increase by about four orders of magnitude to
reach the threshold level required to induce the processes that
lead to lesion formation in tobacco leaves. To prove that the
main biologically active cytokinin increased upon ipt activation
(t-Z) is sufficient to induce lesion formation per se, detached
tobacco leaves were fed with t-Z by dipping their petioles into
t-Z solution in tap water. The effective concentrations of t-Z
start from 1 mM (Supplementary Data Fig. S3).
The increase in cytokinin levels is followed by oxidative stress and
dehydration in expanded leaves
Cytokinins were shown to induce oxidative stress related to
cell death in tobacco BY-2 cells (Mlejnek et al., 2003) and
Downloaded from by guest on March 30, 2015
F I G . 2. Effects of different dexamethasone concentrations on lesion formation in CaMV35S . GR . ipt plants. Five-week-old plants of CaMV35S . GR . ipt line
303 were induced with 50 mL of a dexamethasone solution at the indicated concentrations. The images show plants photographed 91 h after dexamethasone treatment
(4 DAT). Scale bars ¼ 3 cm.
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
(pmol g–1 f. wt)
Hydrogen peroxide
(ratio +DEX/–DEX, %)
(ratio +DEX/–DEX, %)
Leaf dry matter content
(ratio +DEX/–DEX, %)
O -glucosides
(pmol g–1 f. wt)
* *
F I G . 5. Time course of hydrogen peroxide and malondialdehyde accumulation,
and leaf dry matter content in CaMV35S . GR . ipt plants following ipt activation. Plants were treated and samples collected as described in the legend to
Fig. 3. Results are expressed as percentages of the parameters determined in
DEX-treated (+DEX) relative to 0.096 % ethanol-treated (–DEX) plants,
based on the mean values of nine measurements in each case. One and two asterisks indicate statistically significant differences between +DEX and –DEX
plants at P ≤ 0.05 and P ≤ 0.01, respectively, based on the t-test. Error bars indicate s.d.
(pmol g–1 f. wt)
F I G . 4. Time course of cytokinin accumulation in CaMV35S . GR . ipt plants
following ipt activation. Plants were treated and samples collected as described in
the legend to Fig. 3. Cytokinin levels are reported on a logarithmic scale as
the sum of (A) free bases, (B) ribosides and ribotides, (C) O-glucosides and
(D) N9-glucosides. (+) and (–) indicate dexamethasone and 0.096 % ethanol
treatments, respectively. Error bars indicate s.d.
hydrogen peroxide was identified as a major orchestrator of
molecular responses to both biotic and abiotic stresses
(Vandenabeele et al., 2003). Therefore, we examined the hydrogen peroxide levels and membrane damage in leaves that
developed lesions following the activation of the ipt gene. The
potassium iodide assay (Poga´ny et al., 2004) was used to
monitor the levels of hydrogen peroxide in samples of expanded
horizontal leaves. To avoid potential interference arising from
oxidative stresses associated with the degradation processes
taking place in dying cells, only lesion-free plant material was
used in these experiments. Significantly elevated hydrogen peroxide levels were detected in samples harvested 43 h after DEX
application (2 DAT), peaking at 3 DAT (Fig. 5A). The rates and
extent of hydrogen peroxide accumulation in lines 303 and 307
Downloaded from by guest on March 30, 2015
Nucleosides + nucleotides
(pmol g–1 f. wt)
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
The increase in cytokinin levels is followed by downregulation of
photosynthesis-related genes and upregulation of defence-related
Pathogen attacks trigger changes in photosynthetic activity,
while the hypersensitive response involves the induction of
1 cm
1 cm
3 mm
3 mm
F I G . 6. Hydrogen peroxide accumulation in chloroplasts of CaMV35S . GR .
ipt plants following ipt activation. Five-week-old plants of CaMV35S . GR .
ipt line 303 were induced with 50 mL of 20 mM dexamethasone (B, D) or 0.096
% ethanol (A, C) as a control. Leaf samples for histochemical staining using
3,3’-diaminobenzidine were collected 64 h (3 DAT) after treatment with dexamethasone.
pathogenesis-related proteins. To investigate further the similarities between lesion formation resulting from a massive increase
in cytokinin levels and the hypersensitive response caused by
pathogen attack, we examined the expression of photosynthesisand defence-related genes in leaves that developed lesions following ipt activation (Fig. 7). RT – qPCR analysis confirmed a
dramatic increase in ipt transcript abundance 1 DAT. The transcript’s abundance then decreased slightly at 2 DAT and
remained at this slightly lower level 3 DAT. The CAB, FNR1
and VDE genes code for chlorophyll a/b-binding protein,
ferredoxin:NADP oxidoreductase and violaxanthin de-epoxidase, respectively, and were selected as representative
photosynthesis-related genes. In all cases except that of VDE in
line 307, the abundance of the transcripts of these genes fell at
2 DAT. A further decrease (in many cases, to below the limit
of detection) occurred 3 DAT. Once again, VDE in line 307
was exceptional, exhibiting only a mild decrease in transcript
levels 3 DAT relative to the situation 1 DAT. The PR-1b and
PR-Q genes code for pathogenesis-related protein 1b and an
acidic phosphatase, respectively, and were selected as representative pathogenesis-related genes. The abundance of their transcripts began to increase 2 DAT, and rose dramatically over the
following 24 h. In keeping with the relative strength of the leaf
phenotypes in the two lines, the changes in the transcript profiles
were more pronounced in line 303 than in line 307.
Effect of increased cytokinin level on photosynthesis
The gene expression analysis indicated that high cytokinin
levels have inhibitory effects on photosynthesis. To examine
this issue on a physiological level, we analysed a number of
photosynthetic parameters in line 303 following the activation
of the ipt gene. First, the degree of stomatal opening and carboxylation efficiency were determined 67 h after DEX application
(3 DAT; Fig. 8). Light-induced stomatal opening was reduced
significantly in the leaves of plants in which ipt expression was
activated. Microscopic examination revealed that this decreased
stomatal conductance was accompanied by stomatal closure
(data not shown). Similarly, a light-induced increase in the rate
of CO2 assimilation was inhibited, which indicated downregulation of Rubisco activation. The carboxylation efficiency of
Rubisco was also inhibited, as demonstrated by a decrease in
the slope of the linear region of a plot of A against Ci (from
0.025 to 0.015; Fig. 9). We then monitored the changes in chlorophyll fluorescence over time following ipt activation (Figs 10 and
11). A very significant increase in the NPQ occurred within 43 h
of ipt activation (2 DAT) and became slightly more pronounced
over the following 24 h (3 DAT), reflecting an increase in the dissipation of excitation energy. The values of the ETR (II) and ETR
(I) parameters decreased 3 DAT, indicating that electron transport through PSII and PSI was inhibited. There was also an increase in Y(ND), which indicates increased energy dissipation
at the donor side of PSI due to the reduced rate of electron donation to PSI (Klughammer and Schreiber, 2008).
Dynamics of stress hormones following ipt activation in tobacco
ipt activation in lines 303 and 307 triggered a number of processes similar to those observed in the hypersensitive response to
Downloaded from by guest on March 30, 2015
were similar. It thus seems that hydrogen peroxide accumulation
preceded the initiation of lesion formation by at least 7 h.
Histochemical localization of hydrogen peroxide using DAB
staining (Liu et al., 2007) in leaves harvested 3 DAT indicated
that most of the hydrogen peroxide was present in the chloroplasts (Fig. 6). This suggests that the chloroplasts are primarily
responsible for the hydrogen peroxide produced following
increases in cytokinin levels.
To assess the degree of membrane damage caused by
increased hydrogen peroxide levels, we determined the MDA
content of leaf material, which reflects the extent of lipid peroxidation. The time course of MDA accumulation matched that of
hydrogen peroxide in all cases except that at 2 DAT, when the increase in MDA levels was statistically significant only in line 303
(Fig. 5B).
Water losses associated with membrane damage are probably
responsible for the wilting of affected leaves (and in the case of
line 303, their eventual collapse). To quantify this loss, the
LDMC for the two lines was determined (Fig. 5C). Significant
increases in LDMC were observed 3 DAT, i.e. 24 h after the increase in hydrogen peroxide levels. This coincided with a strong
increase in membrane peroxidation. The LDMC in line 303 was
consistently higher than that for line 307, which is consistent with
the much stronger leaf phenotype found in line 303.
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
Normalized expression
1st DAT
Normalized expression
2nd DAT
3rd DAT
Normalized expression
Normalized expression
Normalized expression
F I G . 7. Time course of photosynthesis- and defence-related transcript levels in CaMV35S . GR . ipt plants following ipt activation. Plants were treated and samples
collected as described in Fig. 3. Steady-state transcript levels of (A) genes coding for bacterial isopentenyl transferase (ipt), (B) chlorophyll a/b-binding protein (CAB),
(C) ferredoxin:NADP oxidoreductase (FNR1), (D) violaxanthin de-epoxidase (VDE), (E) pathogenesis-related protein 1b (PR-1b), and (F) acidic phosphatase (PR-Q)
were determined by RT –qPCR using Tob66 and EF-1a transcripts for normalization. Bars indicate s.d.
pathogen attack. Therefore, we examined the dynamics of
pathogenesis- and stress-related hormones following ipt activation (Fig. 12). Pronounced increases in the levels of SA and JA
were observed within 43 h of ipt activation (2 DAT) and these
peaked over the following 24 h at levels that were at least two
orders of magnitude higher than those observed in non-activated
controls. Levels of abscisic acid increased to a lesser degree but
were still between eight and ten times higher than those in the
Effect of decreased stomatal conductance on cell death
The ipt-expressing plants were shown to have strongly reduced
stomatal conductance and thus very limited potential for gas exchange. Such conditions can be simulated by applying lanolin to
the leaf surfaces of non-activated plants (Mateo et al., 2004).
This was done to determine whether the reduced gas-exchange
capabilities of the activated plants could be responsible for the
observed lesion formation. While lanolin treatment did cause
lesion formation, this was only observed when both sides of
the leaves were treated. More importantly, lesion formation
was preceded by chlorosis and it took 3 – 4 weeks of lanolin treatment to achieve a level of lesion formation comparable with that
observed in line 303 only 4 d after ipt activation (Supplementary
Data Fig. S4).
We report a detailed analysis of necrotic lesion formation triggered in tobacco leaves following a dramatic increase in cytokinin levels due to activation of the bacterial cytokinin biosynthesis
gene ipt. Our findings indicate that this process is remarkably
similar to the hypersensitive response in plant –pathogen interactions. We therefore discuss our findings in the context of the potential role of cytokinins in plant – pathogen interactions.
Downloaded from by guest on March 30, 2015
Normalized expression
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
A (mmol CO2 m–2 s–1)
gs (mol H2O m–2 s–1)
y = –1·8 + 0·025x
y = –1·3 + 0·015x
Ci (mmol CO2 mol–1)
F I G . 9. Rate of CO2 assimilation (A) relative to intracellular CO2 concentration
(Ci). Five-week-old plants of CaMV35S . GR . ipt line 303 were induced with
50 mL of 20 mM dexamethasone (+DEX) or 0.096 % ethanol (–DEX) and the A
values at the indicated Ci levels were determined 67 h (3 DAT) after dexamethasone application. Means and the s.d. are shown (n ¼ 3). Dashed lines represent
linear fits for the first four values of the dose–response curve.
Time of actinic illumination (min)
F I G . 8. Light-induced changes in (A) stomatal conductance (gs) and (B) rate of
CO2 assimilation (A). Five-week-old plants of CaMV35S . GR . ipt line 303
were induced with 50 mL of 20 mM dexamethasone (+DEX) or 0.096 %
ethanol (–DEX), and gs and A were determined 67 h (3 DAT) after dexamethasone application. Means and s.d. are shown (n ¼ 3). The photosynthetic photon
flux density of actinic light was 100 mmol photons m22 s21.
ipt activation triggers distinct phenotypes in leaves depending on
their developmental stage
There is a large body of evidence suggesting that cytokinins
are important positive regulators of shoot development (e.g.
Werner et al., 2003; Higuchi et al., 2004; Nishimura et al.,
2004; Riefler et al., 2006). However, we and others have reported
cell death in the leaves of transgenic plants that ectopically
express a gene encoding the key cytokinin biosynthetic
enzyme isopentenyl transferase (Li et al., 2004; Robson et al.,
2004; Sˇa´malova´ et al., 2005; Wingler et al., 2005). While developing and characterizing a stringent and highly responsive
DEX-inducible gene expression system ( pOp6/LhGR) for
tobacco, we observed the development of necrotic lesions,
severe wilting and eventual cell death following ipt activation
in CaMV35S . GR . ipt transgenic tobacco plants (Sˇa´malova´
et al., 2005). Here, we present a detailed analysis of this phenomenon. A single dose of DEX at saturating concentration (20 mM)
caused a dramatic increase in ipt transcription and the size of the
cytokinin pool in 5-week-old transgenic tobacco plants. The best
performing previously characterized systems of inducible ipt expression did not yield levels of free endogenous cytokinin bases
above 0.6 nmol g21 f. wt (Faiss et al., 1997; McKenzie et al.,
1998). In contrast, our system achieved levels of 20 nmol g21 f.
wt at 2 DAT, at which point the cytokinin response was almost
saturated. Whether such high cytokinin levels are produced
under natural conditions in plant tissues remains a challenge
for future work. One can anticipate that high increases in cytokinin levels would be limited to small regions of plant tissue.
Currently, methods allowing reliable determination of plant
hormone metabolites in very small plant samples are being
developed (Nova´k et al., 2012). In this context, it is worth mentioning that some pathogen-derived cytokinins were shown to
increase plant tissue sensitivity even to classical cytokinins
(Pertry et al., 2009).
In shoots, within the first 4 d after ipt activation, clear phenotypic responses were only observed in the leaves, and depended
on their stage of development. Older leaves that had already
achieved a horizontal orientation but were still growing developed necrotic lesions without any prior or accompanying chlorosis, wilted, and eventually died in the more responsive line 303.
Young, upward-oriented leaves developed chlorosis but only
rarely formed lesions. These phenotype changes were observed
only when free cytokinin levels increased by about four orders
of magnitude over their baseline levels. Leaf necrosis associated
with cytokinin overproduction has previously been reported
in leaves of transgenic plants expressing ipt under the control
of senescence-specific promoters. However, in these older
studies, necrosis formation was only observed in old leaves that
were already senescing. In transgenic SAG12-IPT tobacco
plants, cell death was observed only in a fraction of old leaves
and only under conditions of limited nutrient availability
(Wingler et al., 2005). In the more strongly responsive transgenic
maize line Sg3, which carries the Psee1XbaIIPTNOS transgene,
old leaves progressed directly from fully green to bleached and
dead without an intervening yellowing phase, even under conditions of standard nutrient supply (Robson et al., 2004). The
Downloaded from by guest on March 30, 2015
A (µmol CO2 m–2 s–1)
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
303 –DEX
303 +DEX
F I G . 11. False colour images of NPQ in leaves of CaMV35S . GR . ipt plants
following ipt activation. Plants of line 303 were treated as described in
Fig. 3. NPQ was determined under steady-state conditions (10 min of actinic
PAR, 100 mmol photons m22 s21) on 1, 2 and 3 DAT.
Y (ND)
F I G . 10. Time course of chlorophyll fluorescence parameters in CaMV35S .
GR . ipt plants following ipt activation. Plants of line 303 were treated as
described in Fig. 3. The fluorescence parameters (A) NPQ, (B) ETR (II),
(C) ETR (I) and (D) Y(ND) were determined under steady-state conditions
(10 min of actinic PAR, 100 mmol photons m22 s21) 1, 2 and 3 DAT. Means
and s.d. are shown (n ¼ 3– 5). One and two asterisks indicate statistically significant differences between +DEX and –DEX plants at P ≤ 0.05 and P ≤ 0.01, respectively, based on the t-test.
analysis presented herein thus represents the first investigation of
cytokinin-induced necrosis in expanding leaves that have not
undergone any significant reduction in their chlorophyll
content. The responses observed in this work are thus wholly
uncoupled from senescence and are therefore more similar to
the hypersensitive response – a type of cell death associated
with pathogen infection.
In general, cytokinins are considered to be positive regulators
of chloroplast biogenesis and function based on physiological
and anatomical evidence (Chory et al., 1995), as well as transcriptomic (Brenner et al., 2005) and proteomic analyses
(Lochmanova´ et al., 2008; Cˇerny´ et al., 2011). Interestingly,
the predominant cytokinin biosynthesis pathway and some metabolism pathways have been reported to be localized in the
chloroplasts (Brzobohaty´ et al., 1993; Kristoffersen et al.,
2000; Takei et al., 2004; Kiran et al., 2006), and the chloroplast
cytokinin pool has been found to be dynamic (Benkova´ et al.,
1999). Nevertheless, chlorosis has been observed in some transgenic plants that overproduce cytokinins. Decreases in the
chlorophyll content of young leaves similar to those presented
in this work were reported in SAG12-IPT tobacco under conditions of nitrogen remobilization (Jordi et al., 2000), and significant chlorosis in emerging young leaves was found in
Psee1XbaIIPTNOS transgenic maize grown under low nutrient
conditions (Robson et al., 2004). This chlorosis was linked to
defects in the standard remobilization of nutrients from old
leaves, in which senescence is strongly delayed due to a targeted
Downloaded from by guest on March 30, 2015
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
older leaves and/or reductions in the uptake and transport of
mineral nutrients from the soil. In addition to lesion formation,
photosynthesis in older leaves was dramatically inhibited by
increased cytokinin levels by 3 DAT. Growth of the root
system decreased by 25 % in plants with an increased cytokinin
level 4 DAT. In roots, cytokinins were reportedly shown to affect
negatively the uptake of mineral nutrients including nitrate, ammonium, phosphate and iron (reviewed in Rubio et al., 2009).
This is consistent with the observation that chlorosis only
became apparent 4 DAT. As a matter of fact, there was a slight
increase in chlorophyll levels 2 DAT in the young leaves of the
less responsive line 307, although they declined later on.
Nevertheless, our data cannot exclude that increased cytokinin
levels per se caused the chlorosis in young leaves directly.
Salicylic acid
(pmol g–1 f. wt)
The hypersensitive-like response triggered by increased cytokinin
Abscisic acid
(pmol g–1 f. wt)
F I G . 12. Time course of accumulation of stress-related hormones in
CaMV35S . GR . ipt plants following ipt activation. Plants were treated as
described in Fig. 3, and samples for (A) salicylic, (B) jasmonic and (C) abscisic
acid analysis were collected at 19 h (1 DAT), 43 h (2 DAT), 67 h (3 DAT) and 91 h
(4 DAT) of dexamethasone application. Hormone levels are presented on a logarithmic scale. (+) and (–) indicate dexamethasone and 0.096 % ethanol treatment, respectively. Error bars are s.d.
increase in cytokinin levels. Thus, chlorosis reported previously
in ipt-expressing plants is unlikely to result directly from the
effects of cytokinins in young leaves. Similarly, the chlorosis
observed in young leaves in this work might result from
decreased assimilate availability due to extensive damage in
Cytokinins reportedly play distinct roles in plant – pathogen
interactions (reviewed by Argueso et al., 2009; Choi et al.,
2011) which are dependent, at least in part, on the nature of the
pathogen. While necrotrophs kill the cells of the host plant, biotrophs cause minimal cellular damage and require living host
tissues. They can produce cytokinins and auxins to modulate
the physiology of host plants to support biotroph proliferation.
On the other hand, plant derived-cytokinins are reportedly
involved in plant resistance to viral infection and other pathogens
that do not secrete cytokinins. For example, cytokinins were
shown to modulate SA signalling to increase resistance against
Pseudomonas syringae in Arabidopsis (Choi et al., 2010).
Infected plants can activate localized programmed cell death
related to the hypersensitive response which impairs pathogen
spread. The hypersensitive response is induced by ROS and reactive nitrogen species. Cytokinins were shown to induce programmed cell death (Mlejnek and Procha´zka, 2002; Carimi
et al., 2003); however, their involvement in the hypersensitive response has not been reported.
Here we show that the dramatic increase in cytokinin levels
triggered by ipt activation resulted in extensive cell death and
lesion formation in older leaves. Since this was not accompanied
or preceded by chlorosis, the overall process closely resembled
the hypersensitive response associated with pathogen attack.
The hypersensitive-like nature of the cytokinin response is
further demonstrated by the observed changes in various biochemical and molecular parameters prior to the appearance of
the first islets of dead cells in affected leaves. One of the early
consequences of increased cytokinin levels identified in this
work was an increase in the levels of chloroplast-associated
hydrogen peroxide. This was accompanied by oxidative membrane damage, increases in stress hormone levels and PR transcripts, and decreases in the abundance of transcripts related to
photosynthesis. High doses of exogenous cytokinin have also
been reported to increase the levels of ROS in tobacco BY2
cells (Mlejnek et al., 2003). Our data provide the first evidence
for a chloroplast-associated burst of hydrogen peroxide production in response to endogenously produced tZ-type cytokinins
in planta. Since milder increases in cytokinin levels have been
reported to stimulate enzymes involved in ROS detoxification
(Zavaleta-Mancera et al., 2007; Barna et al., 2008), our data
suggest that cytokinins have two separate and concentration-
Downloaded from by guest on March 30, 2015
Jasmonic acid
(pmol g–1 f. wt)
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
Inhibition of photosynthesis upon ipt activation in transgenic
Photosynthesis, ROS levels and the hypersensitive response
are intertwined at several levels (for a recent review, see
Kangasja¨rvi et al., 2012). It has been argued that chloroplastic
ROS production contributes to plant immunity (Karpinski
et al., 1999; Vandenabeele et al., 2004), and photosynthetic electron transfer reactions are known to be a significant source of
ROS (Hideg et al., 2002). In addition, the photoactive nature of
chlorophyll provides another mechanism for ROS formation in
chloroplasts (Lorrain et al., 2003). However, photosynthesis is
inhibited by high levels of ROS (Vandenabeele et al., 2003).
We observed downregulation of CAB, FNR1 and VDE transcripts
related to photosynthesis within 43 h of ipt activation, which
might indicate a reduction in photosynthetic activity. A similar
downregulation of the corresponding genes was observed following the activation of the ipt gene in Arabidopsis (Hoth
et al., 2003). Together with the fact that chlorophyll levels
remained unchanged, the downregulation of CAB transcript
levels might indicate the presence of free chlorophyll in the
chloroplasts, which could contribute to ROS generation in the
tobacco leaves that developed lesions following ipt activation.
Analyses of chlorophyll fluorescence and CO2 assimilation
revealed that photosynthetic activity declined following ipt activation. Moreover, electron transport through PSII and PSI was
inhibited, there was an increase in the amount of energy dissipated at the donor side of PSI due to reduced electron donation
and there was an increase in the dissipation of excitation
energy by heat emission, as demonstrated by an increase in
NPQ. The need to dissipate excess chlorophyll excitation
energy via NPQ reflects a decrease in electron consumption by
CO2 assimilation resulting from the downregulation of Rubisco
activity, together with a decrease in CO2 availability due to
reduced stomatal conductance. Decreases in CO2 assimilation
reportedly lead to an excess of excitation energy, the formation
of ROS and hypersensitive-like cell death (Mateo et al., 2004;
Liu et al., 2007). However, in our system, the decrease in CO2
availability did not seem to be the main contributor to the
hypersensitive-like response induced by cytokinins since reductions in CO2 availability following treatment with lanolin paste
(Mateo et al., 2004) were not by themselves sufficient to
trigger this response on a time scale of 4 d. The decrease in stomatal conductivity correlated with closure of the stomata.
Cytokinins are generally considered to promote stomatal
opening and decrease sensitivity to ABA. However, the
responses to hormones are often dependent on their abundance
(reviewed by Acharya and Assmann, 2009). We found a good
correlation between stomatal closure and the kinetics of the increase in ABA levels. We have previously observed elevated
ABA levels in tobacco plants overexpressing the Sho gene,
which codes for a cytokinin biosynthetic enzyme from Petunia
hybrida (Polanska´ et al., 2007). The occurrence of cross-talk
between cytokinin and ABA on the metabolic level is further
supported by findings such as those obtained in an analysis of
transgenic tobacco overexpressing Arabidopsis cytokinin
oxidase/dehydrogenase AtCKX3. In these plants, the decrease
in cytokinin levels caused by AtCKX3 activity was accompanied
by a decrease in ABA content (Polanska´ et al., 2007). It therefore
seems that an increase in the levels of ABA and hydrogen peroxide is probably responsible for the observed stomatal closure following ipt activation in CaMV35S . GR . ipt tobacco plants.
The stomatal closure probably represents an insufficient
attempt to prevent the water losses that accompany elevated cytokinin levels. However, stomatal closure also protects the plant
against pathogen invasion (Melotto et al., 2006) and so would
be an expected response to pathogen-related hormones.
Dramatic increases in endogenous cytokinin levels triggered
by activating the ipt gene from the bacterial soil plant pathogen
A. tumefaciens cause a hypersensitive-like response in transgenic tobacco leaves. The data presented herein are consistent with
the suggestion that the molecular processes underpinning this
hypersensitive-like response are orchestrated by increases in cellular hydrogen peroxide levels. The hypersensitive-like response
includes inhibition of photosynthesis, increases in stress
Downloaded from by guest on March 30, 2015
dependent effects on the cellular ROS pool. Hydrogen peroxide
has been shown to orchestrate molecular responses to both biotic
and abiotic stresses, and transcriptomic analysis demonstrated
that hydrogen peroxide production is closely linked to the
levels of stress hormones such as SA, JA and ethylene
(Vandenabeele et al., 2003). Salicylic acid is important in
plants’ defences against pathogens: it can prevent local infections and also mediate systemic acquired resistance, promoting
resistance at sites remote from the point of infection. Jasmonic
acid mediates local and systemic defence responses to wounding
by herbivores. Cytokinins were reported to modulate SA signalling to enhance resistance against P. syringae (Choi et al., 2010),
and a systems analysis revealed a synergism between cytokinin
and SA in plant disease networks (Naseem et al., 2012).
However, direct stimulation of either SA or JA accumulation in
response to cytokinin action has not been documented. On the
contrary, SA levels decreased relative to those in an untransformed control in transgenic tobacco explants that were propagated in vitro and overproduced cytokinins due to ipt
expression driven by a promoter of the Rubisco small subunit
(Schnablova´ et al., 2006). In poplars, treatment with exogenous
cytokinins did not affect JA levels in undamaged leaves.
However, cytokinin priming increased the wound-inducible accumulation of JA during insect attacks on the poplar leaves
(Dervinis et al., 2010). It thus seems that hydrogen peroxide
mediated the increase in the levels of SA and JA that was
observed in this work following increases in cytokinin abundance. The induction of PR protein expression is a key component of local and systemic defence responses. The
transcriptional activation of many PR genes is regulated by an
SA-mediated signal transduction cascade (reviewed by Van
Loon, 1997), and a similar system may be responsible for the
transcriptional activation observed in this work. On the other
hand, a number of PR proteins are encoded by late cytokinin response genes (Rashotte et al., 2003). It is therefore possible that
the activation of the PR-1b and PR-Q genes observed in this work
could be a direct consequence of cytokinin signalling.
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
hormone levels, oxidative damage of membranes and stomatal
closure. Overall, the data indicate that cytokinins can act as mediators in plant – pathogen interactions. This conclusion is consistent with the results of recent dynamic modelling studies and
systems analyses, which identified multiple cytokinin-mediated
regulatory interactions in plant disease networks (Naseem et al.,
We thank Dr Ian Moore for CaMV35S.GR.ipt seeds. We
thank Petra Amakorova´ and Hana Martı´nkova´ for great technical
assistance. This work was supported by grant nos 1M06030 and
LK21306 (Ministry of Education, Youth and Sports of the Czech
Republic), and projects ‘CEITEC –Central European Institute of
Technology’ (CZ.1.05/1.1.00/02.0068) and ‘Centre of the
Region Hana´ for Biotechnological and Agricultural Research’
(grant no. ED0007/01/01) from the European Regional
Developmental Fund. Access to the Meta-Centrum computing
facilities provided under the programme ‘Projects of Large
Infrastructure for Research, Development, and Innovations’
(LM2010005), which is funded by the Ministry of Education,
Youth and Sports of the Czech Republic, is highly appreciated.
Acharya BR, Assmann SM. 2009. Hormone interactions in stomatal function.
Plant Molecular Biology 69: 451–462.
Alexieva V, Sergiev I, Mapelli S, Karanov E. 2001. The effect of drought and
ultraviolet radiation on growth and stress markers in pea and wheat. Plant,
Cell and Environment 24: 1337–1344.
Argueso CT, Ferreira FJ, Kieber JJ. 2009. Environmental perception avenues:
the interaction of cytokinin and environmental response pathways. Plant,
Cell and Environment 32: 1147–1160.
Ashikari M, Sakakibara H, Lin S, et al. 2005. Cytokinin oxidase regulates rice
grain production. Science 309: 741– 745.
Barna B, Smigocki C, Baker JC. 2008. Transgenic production of cytokinin suppresses bacterially induced hypersensitive response symptoms and
increases antioxidative enzyme levels in Nicotiana spp. Phytopathology
98: 1242– 1247.
Benkova´ E, Witters E, Van Dongen W, et al. 1999. Cytokinins in tobacco and
wheat chloroplasts: occurrence and changes due to light/dark treatment.
Plant Physiology 121: 245–252.
Bergougnoux V, Hlava´cˇkova´ V, Plotzova´ R, Nova´k O, Fellner M. 2009. The
7B-1 mutation in tomato (Solanum lycopersicum L.) confers a blue lightspecific lower sensitivity to coronatine, a toxin produced by Pseudomonas
syringae pv. tomato. Journal of Experimental Botany 60: 1219–1230.
Brenner WG, Romanov GA, Ko¨llmer I, Bu¨rkle L, Schmu¨lling T. 2005.
Immediate-early and delayed cytokinin response genes of Arabidopsis thaliana identified by genome-wide expression profiling reveal novel cytokininsensitive processes and suggest cytokinin action through transcriptional cascades. The Plant Journal 44: 314 –333.
Brzobohaty´ B, Moore I, Kristoffersen P, et al. 1993. Release of active
cytokinin by a b-glucosidase localized to the maize root meristem.
Science 262: 1051– 1054.
von Caemmerer S, Farquhar GD. 1981. Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 154:
Carimi F, Zottini M, Formentin E, Terzi M, Lo Schiavo F. 2003. Cytokinins:
new apoptotic inducers in plants. Planta 216: 413–421.
Chilton MD, Drummond MH, Merio DJ, et al. 1977. Stable incorporation of
plasmid DNA into higher plant cells: the molecular basis of crown gall
tumorigenesis. Cell 11: 263–271.
Choi J, Huh SU, Kojima M, Sakakibara H, Paek K-H, Hwang I. 2010. The
cytokinin-activated transcription factor ARR2 promotes plant immunity
via TGA3/NPR1-dependent salicylic acid signaling in Arabidopsis.
Developmental Cell 19: 284–295.
Choi J, Choi D, Lee S, Ryu C-M, Hwang I. 2011. Cytokinins and plant immunity: old foes or new friends? Trends in Plant Science 16: 388– 394.
Clarke SF, Burritt DJ, Jameson PE, Guy PL. 1998. Influence of plant hormones on virus replication and pathogenesis-related proteins in Phaseolus
vulgaris L. infected with white clover mosaic potexvirus. Physiological
and Molecular Plant Pathology 53: 195–207.
Clarke S, McKenzie M, Burritt D, Guy P, Jameson P. 1999. Influence of
white clover mosaic potexvirus infection on the endogenous cytokinin
content of bean. Plant Physiology 120: 547–552.
ˇ erny´ M, Dycˇka F, Boba´l’ova´ J, Brzobohaty´ B. 2011. Early cytokinin response
proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling. Journal of Experimental Botany 62:
Dangl JL, Dietrich RA, Richberg MH. 1996. Death don’t have no mercy:
cell death programs in plant– microbe interactions. The Plant Cell 8:
1793– 1807.
Dervinis C, Frost CJ, Lawrence SD, Novak NG, Davis JM. 2010. Cytokinin
primes plant responses to wounding and reduces insect performance.
Journal of Plant Growth Regulation 29: 289–296.
Devos S, Laukens K, Deckers P, et al. 2006. A hormone and proteome approach
to picturing the initial metabolic events during Plasmodiophora brassicae
infection on Arabidopsis. Molecular Plant-Microbe Interactions 19:
1431– 1443.
Faiss M, Zalubilova´ J, Strnad M, Schmu¨lling T. 1997. Conditional transgenic
expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants. The Plant Journal 12: 401–415.
Gan S, Amasino RM. 1995. Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270: 1986–1988.
Gonzalez-Rizzo S, Crespi M, Frugier F. 2006. The Medicago truncatula CRE1
cytokinin receptor regulates lateral root development and early symbiotic
interaction with Sinorhizobium meliloti. The Plant Cell 18: 2680– 2693.
Ha S, Vanˇkova´ R, Yamaguchi-Shinozaki K, Shinozaki K, Tran LS. 2012.
Cytokinins: metabolism and function in plant adaptation to environmental
stresses. Trends in Plant Science 17: 172–179.
Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts.
I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of
Biochemistry and Biophysics 125: 189–198.
Hideg E, Barta C, Kalai T, Vass I, Hideg K, Asada K. 2002. Detection of
singlet oxygen and superoxide with fluorescent sensors in leaves under
stress by photoinhibition or UV radiation. Plant and Cell Physiology 43:
1154– 1164.
Higuchi M, Pischke M, Ma¨ho¨nen AP, et al. 2004. In planta functions of the
Arabidopsis cytokinin receptor family. Proceedings of the National
Academy of Sciences, USA 101: 8821–8826.
Hoth S, Ikeda Y, Morgante M, et al. 2003. Monitoring genome-wide changes in
gene expression in response to endogenous cytokinin reveals targets in
Arabidopsis thaliana. FEBS Letters 554: 373– 380.
Jordi W, Schapendonk A, Davelaar E, et al. 2000. Increased cytokinin levels
in transgenic PSAG12-IPT tobacco plants have large direct and indirect
effects on leaf senescence, photosynthesis and N partitioning. Plant, Cell
and Environment 23: 279–289.
Downloaded from by guest on March 30, 2015
Supplementary data are available online at and consist of the following. Table S1: primers
and amplification conditions used for gene expression analysis.
Table S2: time course of cytokinin accumulation in
CaMV35S . GR . ipt plants following ipt activation. Figure
S1: time course of chlorophyll content in older leaves of
CaMV35S . GR . ipt plants following ipt activation. Figure
S2: cytokinin content of CaMV35S . GR . ipt plants following ipt activation. Figure S3: effects of different t-Z concentrations on lesion formation in detached leaves of wild-type
plants. Figure S4: effects of lanolin on lesion formation in
tobacco leaves.
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
single-quadrupole mass spectrometry. Analytica Chimica Acta 480:
207– 218.
Nova´k O, Hauserova´ E, Amakorova´ P, Dolezˇal K, Strnad M. 2008.
Cytokinin profiling in plant tissues using ultra-performance liquid chromatography – electrospray tandem mass spectrometry. Phytochemistry
69: 2214 – 2224.
Nova´k O, Henykova´ E, Sairanen I, Kowalczyk M, Pospı´sˇil T, Ljung K. 2012.
Tissue-specific profiling of the Arabidopsis thaliana auxin metabolome.
The Plant Journal 72: 523–536.
O’Brien JA, Daudi A, Finch P, et al. 2012. A peroxidase-dependent
apoplastic oxidative burst in cultured Arabidopsis cells functions in
MAMP-elicited defense. Plant Physiology 158: 2013– 2027.
Pertry I, Va´clavı´kova´ K, Depuydt S, et al. 2009. Identification of Rhodococcus
fascians cytokinins and their modus operandi to reshape the plant.
Proceedings of the National Academy of Sciences, USA 106: 929– 934.
Poga´ny M, Koehl J, Heiser I, Elstner E, Barna B. 2004. Juvenility of tobacco
induced by cytokinin gene introduction decreases susceptibility to and
confers tolerance to oxidative stress. Physiological and Molecular Plant
Pathology 65: 39–47.
Polanska´ L, Vicˇa´nkova´ A, Nova´kova´ M, et al. 2007. Altered cytokinin metabolism affects cytokinin, auxin, and abscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure in transgenic tobacco. Journal of
Experimental Botany 58: 637– 649.
Prokopova´ J, Sˇpundova´ M, Sedla´rˇova´ M, et al. 2010. Photosynthetic responses
of lettuce to downy mildew infection and cytokinin treatment. Plant
Physiology and Biochemistry 48: 716– 723.
Rashotte AM, Carson SDB, To JPC, Kieber JJ. 2003. Expression profiling of cytokinin action in Arabidopsis. Plant Physiology 132:
1998 – 2011.
Riefler M, Nova´k O, Strnad M, Schmu¨lling T. 2006. Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size,
germination, root development, and cytokinin metabolism. The Plant Cell
18: 40–54.
Rivero RM, Kojima M, Gepstein A, et al. 2007. Delayed leaf senescence
induces extreme drought tolerance in a flowering plant. Proceedings of the
National Academy of Sciences, USA 104: 19631–19636.
Robson PRH, Donnison IS, Wang K, et al. 2004. Leaf senescence is delayed in
maize expressing the Agrobacterium IPT gene under the control of a novel
maize senescence-enhanced promoter. Plant Biotechnology Journal 2:
101– 112.
Rubio V, Bustos R, Irigoyen ML, Cardona-Lo´pez X, Rojas-Triana M,
Paz-Ares J. 2009. Plant hormones and nutrient signaling. Plant
Molecular Biology 69: 361– 373.
Rushton PJ, Somssich IE. 1998. Transcriptional control of plant genes responsive to pathogens. Current Opinion in Plant Biology 1: 311–315.
Sˇa´malova´ M, Brzobohaty´ B, Moore I. 2005. pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression
system for tobacco. The Plant Journal 41: 919– 935.
Schnablova´ R, Synkova´ H, Vica´nkova´ A, Burketova´ L, Eder J, Cvikrova´ M.
2006. Transgenic ipt tobacco overproducing cytokinins overaccumulates
phenolic compounds during in vitro growth. Plant Physiology and
Biochemistry 44: 526–534.
Siemens J, Keller I, Sarx J, et al. 2006. Transcriptome analysis of Arabidopsis
clubroots indicate a key role for cytokinins in disease development.
Molecular Plant-Microbe Interactions 19: 480– 494.
Takei K, Ueda N, Aoki K, et al. 2004. AtIPT3 is a key determinant of nitratedependent cytokinin biosynthesis in Arabidopsis. Plant and Cell
Physiology 45: 1053–1062.
Tirichine L, Sandal N, Madsen LH, et al. 2007. A gain-of-function mutation in
a cytokinin receptor triggers spontaneous root nodule organogenesis.
Science 5: 104– 107.
Vandenabeele S, Van Der Kelen K, Dat J, et al. 2003. A comprehensive analysis of hydrogen peroxide-induced gene expression in tobacco.
Proceedings of the National Academy of Sciences, USA 100: 16113–16118.
Vandenabeele S, Vanderauwera S, Vuylsteke M, et al. 2004. Catalase deficiency drastically affects gene expression induced by high light in
Arabidopsis thaliana. The Plant Journal 39: 45–58.
Vandesompele J, De Preter K, Pattyn F, et al. 2002. Accurate normalization of
real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3: research0034-research0034.11.
Downloaded from by guest on March 30, 2015
Kangasja¨rvi S, Neukermans J, Li S, Aro E-M, Noctor G. 2012.
Photosynthesis, photorespiration, and light signalling in defence responses.
Journal of Experimental Botany 63: 1619–1636.
Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux
P. 1999. Systemic signalling and acclimation in response to excess excitation energy in Arabidopsis. Science 284: 654–657.
Kiran NS, Polanska´ L, Fohlerova´ R, et al. 2006. Ectopic over-expression of the
maize b-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco. Journal of Experimental Botany 57: 985–996.
Klughammer C, Schreiber U. 2008. Saturation pulse method for assessment of
energy conversion in PSI. PAM Application Notes 1: 11–14.
Kristoffersen P, Brzobohaty´ B, Ho¨hfeld I, Bako L, Melkonian M, Palme K.
2000. Developmental regulation of the maize Zm-p60.1 gene encoding a
b-glucosidase located to plastids. Planta 210: 407 –415.
Li Q, Robson PRH, Bettany AJE, Donnison IS, Thomas H, Scott IM. 2004.
Modification of senescence in ryegrass transformed with IPT under the
control of a monocot senescence-enhanced promoter. Plant Cell Reports
22: 816– 821.
Liu Y, Ren D, Pike S, Pallardy S, Gassmann W, Zhang S. 2007.
Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein
kinase cascade. The Plant Journal 51: 941– 954.
Lochmanova´ G, Zdra´hal Z, Konecˇna´ H, et al. 2008. Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis. Journal of
Experimental Botany 59: 3705– 3719.
Lorrain S, Vailleau F, Balague´ C, Roby D. 2003. Lesion mimic mutants: keys
for deciphering cell death and defense pathways in plants? Trends in Plant
Science 8: 263–271.
Love AJ, Milner JJ, Sadanandom A. 2008. Timing is everything: regulatory
overlap in plant cell death. Trends in Plant Science 13: 589–595.
Masferrer A, Arro´ M, Manzano D, et al. 2002. Overexpression of Arabidopsis
thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis
induces a cell death/senescence-like response and reduced cytokinin
levels. The Plant Journal 30: 123– 132.
Mateo A, Muhlenbock P, Rusterucci C, et al. 2004. LESION SIMULATING
DISEASE 1 is required for acclimation to conditions that promote excess excitation energy. Plant Physiology 136: 2818– 2830.
McCabe MS, Garratt LC, Schepers F, et al. 2001. Effects of PSAG12-IPT gene
expression on development and senescence in transgenic lettuce. Plant
Physiology 127: 505– 516.
McKenzie M, Mett V, Reynolds PHS, Jameson P. 1998. Controlled cytokinin
production in transgenic tobacco using a copper-inducible promoter. Plant
Physiology 116: 969– 977.
Melotto M, Underwood W, Koczan J, Nomura K, He SY. 2006. Plant stomata
function in innate immunity against bacterial invasion. Cell 126: 969–980.
Miller CO, Skoog F, Von Saltza MH, Strong F. 1955. Kinetin, a cell division
factor from deoxyribonucleic acid. Journal of the American Chemical
Society 77: 1392.
Mlejnek P, Procha´zka S. 2002. Activation of caspase-like proteases and induction of apoptosis by isopentenyladenosine in tobacco BY-2 cells. Planta
215: 158–166.
Mlejnek P, Dolezˇel P, Procha´zka S. 2003. Intracellular phosphorylation of benzyladenosine is related to apoptosis induction in tobacco BY-2 cells. Plant,
Cell and Environment 26: 1723– 1735.
Mlejnek P, Dolezˇel P, Procha´zka S. 2005. Intracellular conversion of cytokinin
bases into corresponding mononucleotides is related to cell death induction
in tobacco BY-2 cells. Plant Science 168: 389– 395.
Murray JD, Karas BJ, Sato S, Tabata S, Amyot L, Szczyglowski K. 2006.
A cytokinin perception mutant colonized by Rhizobium in the absence of
nodule organogenesis. Science 5: 101 –104.
Naseem M, Philippi N, Hussain A, Wangorsch G, Ahmed N, Dendakar T.
2012. Integrated systems view on networking by hormones in Arabidopsis
immunity reveals multiple crosstalk for cytokinin. The Plant Cell 24:
Nishimura C, Ohashi Y, Sato S, Kato T, Tabata S, Ueguchi C. 2004. Histidine
kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. The Plant
Cell 16: 1365– 1377.
Nova´k O, Tarkowski P, Tarkowska´ D, Dolezˇal K, Lenobel R, Strnad M. 2003.
Quantitative analysis of cytokinins in plants by liquid chromatography/
Nova´k et al. — Cytokinins can trigger hypersensitive-like response
Van Loon LC. 1997. Induced resistance in plants and the role of pathogenesisrelated proteins. European Journal of Plant Pathology 103: 753 –765.
Wellburn AR. 1994. The spectral determination of chlorophylls a and b, as well
as total carotenoids, using various solvents with spectrophotometers of different resolution. Journal of Plant Physiology 144: 307–313.
Werner T, Schmu¨lling T. 2009. Cytokinin action in plant development. Current
Opinion in Plant Biology 12: 527– 538.
Werner T, Motyka V, Laucou V, Smets R, Onckelen HV, Schmu¨lling T. 2003.
Cytokinin-deficient transgenic Arabidopsis plants show functions of
cytokinins in the regulation of shoot and root meristem activity. The Plant
Cell 15: 2532– 2550.
Wingler A, Brownhill E, Pourtau N. 2005. Mechanisms of the light-dependent
induction of cell death in tobacco plants with delayed senescence. Journal of
Experimental Botany 56: 2897– 2905.
Zavaleta-Mancera HA, Lo´pez-Delgado H, Loza-Tavera H, et al. 2007.
Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence. Journal of Plant
Physiology 164: 1572–1582.
Downloaded from by guest on March 30, 2015

High cytokinin levels induce a hypersensitive-like