Journal of Experimental Botany, Vol. 56, No. 421, pp. 2877–2883, November 2005
doi:10.1093/jxb/eri282 Advance Access publication 12 September, 2005
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Diurnal variation of cytokinin, auxin and abscisic acid
levels in tobacco leaves
Marie Nováková1,2, Václav Motyka1, Petre I. Dobrev1, Jiřı́ Malbeck1, Alena Gaudinová1
and Radomı́ra Vanková1,*
1
Institute of Experimental Botany AS CR, Rozvojova 135, 165 02 Prague 6, Czech Republic
2
Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 43 Prague 2,
Czech Republic
Received 15 April 2005; Accepted 5 August 2005
As many processes are regulated by both light and
plant hormones, evaluation of diurnal variations of
their levels may contribute to the elucidation of the
complex network of light and hormone signal transduction pathways. Diurnal variation of cytokinin, auxin,
and abscisic acid levels was tested in tobacco leaves
(Nicotiana tabacum L. cv. Wisconsin 38) grown under
a 16/8 h photoperiod. The main peak of physiologically
active cytokinins (cytokinin bases and ribosides) was
found after 9 h of light, i.e. 1 h after the middle of the
light period. This peak coincided with the major auxin
peak and was closely followed by a minor peak of
abscisic acid. Free abscisic acid started to increase at
the light/dark transition and reached its maximum 3 h
after dark initiation. The content of total cytokinins
(mainly N-glucosides, followed by cis-zeatin derivatives and nucleotides) exhibited the main peak after
9 h of light and the minor peak after the transition to
darkness. The main, midday peak of active cytokinins
was preceded by a period of minimal metabolic conversion of tritiated trans-zeatin (less than 30%). The
major cytokinin-degrading enzyme, cytokinin oxidase/
dehydrogenase (EC 1.5.99.12), exhibited maximal activity after the dark/light transition and during the
diminishing of the midday cytokinin peak. The former
peak might be connected with the elimination of the
long-distance cytokinin signal. These cytokinin oxidase/dehydrogenase peaks were accompanied by increased activity of b-glucosidase (EC 3.2.1.21), which
might be involved in the hydrolysis of cytokinin
O-glucosides and/or in fine-tuning of active cytokinin levels at their midday peak. The achieved data indicate that cytokinin metabolism is tightly regulated
by the circadian clock.
Key words: Abscisic acid, auxin, cytokinin, cytokinin oxidase/
dehydrogenase, diurnal rhythm, b-glucosidase, tobacco.
Introduction
It is assumed that plant growth and development are
regulated by the interactions between the environment
and endogenous factors, especially hormones. Light is an
important environmental signal that activates and modifies
various growth and developmental processes in plants, for
example, seed germination, flower induction, leaf movements, stem elongation, and de-etiolation. All of these
processes are regulated by plant hormones as well. Effects
of light and phytohormones can be synergistic (e.g.
chloroplast development and cytokinins [CKs]), additive
(e.g. inhibition of hypocotyls elongation and CKs), or
antagonistic (e.g. germination and abscisic acid [ABA]).
There are numerous reports that light itself may influence
the levels of different hormones. Light was found to
regulate directly the biosynthesis of active gibberellins
(Foster and Morgan, 1995) and ethylene (Jasoni et al.,
2002) as well as to promote the degradation of ABA
(Kraepiel et al., 1994). The effect of light on auxin levels is
less transparent and may be realized by influencing auxin
* To whom correspondence should be addressed. Fax: +420 220 390 446. E-mail: vankova@ueb.cas.cz
Abbreviations: ABA, abscisic acid; CK, cytokinin; CKX, cytokinin oxidase/dehydrogenase; DHZ, dihydrozeatin; DHZR, dihydrozeatin riboside; IAA, b-indolylacetic acid; iP, N 6-(D2-isopentenyl)adenine; iPR, N 6-(D2-isopentenyl)adenosine; iP7G, N6-(D2-isopentenyl)adenine 7-glucoside; iP9G, N6-(D2-isopentenyl)
adenine 9-glucoside; Z, trans-zeatin; ZR, trans-zeatin riboside; ZOG, trans-zeatin O-glucoside; ZROG, trans-zeatin riboside O-glucoside; Z7G, trans-zeatin
7-glucoside; Z9G, trans-zeatin 9-glucoside.
ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
The online version of this article has been published under an Open Access model. Users are entitled to use, reproduce, disseminate, or display the Open Access version of this article
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Abstract
2878 Nováková et al.
Materials and methods
Plant material
Tobacco plants (Nicotiana tabacum L. cv. Wisconsin 38) were grown
in soil in a growth chamber for 6 weeks at a 16 h photoperiod and
130 lmol mÿ2 sÿ1, a day/night temperature of 25/23 8C, and a relative
humidity of c. 80%. Mixed samples of leaves (3rd–5th of 7, i.e. with
the exception of the youngest and oldest ones) from three plants were
collected every hour during a 24 h period. After removal of the main
vein the samples were immediately frozen in liquid nitrogen.
Incubation with tritiated trans-zeatin
Tip parts of the 3rd or 4th leaf (c. 1 g FW) were cut at 3 h intervals and
their cut surface was submerged into aqueous tritiated trans-zeatin
([3H]Z, 33104 Bq, specific activity 0.6 TBq mmolÿ1, prepared by
Dr Jan Hanuš, Isotope Laboratory, Institute of Experimental Botany
AS CR, Prague, Czech Republic). After 1 h incubation (under the
same light regime as the intact plants) leaf tips were frozen in liquid
nitrogen. The uptake of [3H]Z by the vascular tissue was efficient
(35–50% of the applied radioactivity).
Plant hormone extraction and purification
Phytohormones were extracted and purified according to Dobrev and
Kamı́nek (2002). Frozen leaf samples (c. 1 g FW) were ground in
liquid nitrogen and extracted overnight with 10 cm3 methanol/water/
formic acid (15/4/1, by vol., pH;2.5, ÿ20 8C). For analyses of
endogenous CKs, 50 pmol of each of the following 12 deuteriumlabelled standards were added: [2H5]Z, [2H5]ZR, [2H5]Z7G,
[2H5]Z9G, [2H5]ZOG, [2H5]ZROG, [2H6]iP, [2H6]iPR, [2H6]iP7G,
[2H6]iP9G, [2H5]DHZ, [2H5]DHZR (Apex Organics, Honington,
UK). Tritiated internal standards were used for the determination of
auxin (3[5(n)-3H] indolyl acetic acid, Amersham, UK, specific
activity 74 GBq mmolÿ1, 53103 Bq) and ABA (Amersham, UK,
specific activity 1.74 TBq mmolÿ1, 53103 Bq). The extracts were
purified using Si-C18 columns (SepPak Plus, Waters, Milford, MC,
USA) and Oasis MCX mixed mode (cation exchange and reversephase) columns (150 mg, Waters, USA) and evaporated.
HPLC of radiolabelled trans-zeatin and its metabolic products
Evaporated samples were resuspended in 200 mm3 20% (v/v)
acetonitrile. Radiolabelled metabolites of [3H]Z were analysed
according to Blagoeva et al. (2004) using a Series 200 Quaternary
HPLC Pump (Perkin Elmer, Wellesley, MA, USA) coupled to a
235C Diode Array Detector (Perkin Elmer, USA) and a RAMONA
2000 flow-through radioactivity detector (Raytest, Straubenhardt,
Germany), column: Luna C18(2), 150 mm/4.6 mm/3 lm (Phenomenex, Torrance, CA, USA). The radioactive metabolites were identified on the basis of coincidence of retention times with authentic
standards.
HPLC of auxin and abscisic acid
Indolyl acetic acid and ABA were determined using two-dimensional
HPLC according to Dobrev et al. (2005). The instrumental set-up
consisted of a series 200 autosampler (Perkin Elmer, Norwalk, CT,
USA), two HPLC gradient pump systems (first pump: ConstaMetric
3500 and 3200 with 500 ll mixer, TSP, Riviera Beach, FL, USA;
second pump: Series 200 Quaternary Pump, Perkin Elmer), two
columns (first column: ACE-3CN, 15034.6 mm, 3 mm, ACT,
Aberdeen, Scotland, UK; second column: Luna C18(2), 15034.6
mm, 3 mm, Phenomenex, Torrance, CA, USA), one 2-position, fluid
processor SelectPRO with 1 ml loop (Alltech, Deerfield, IL, USA).
The segment containing IAA and ABA obtained in the first dimension was collected in the loop of the fluid processor and
redirected to the second HPLC dimension. IAA was quantified
using fluorescence detector LC 240 (Perkin Elmer). Quantification
of ABA was performed on the basis of UV detection using diode
array-detector 235C (Perkin Elmer).
HPLC/mass spectrometry
LC-MS analysis was performed as described by Lexa et al. (2003)
using a Rheos 2000 HPLC gradient pump (Flux Instruments,
Switzerland) and HTS PAL autosampler (CTC Analytics, Switzerland) coupled to an Ion Trap Mass Spectrometer Finnigan MAT
LCQ-MSn equipped with an electrospray interface. Detection and
quantification were carried out using a Finnigan LCQ operated in the
positive ion, full-scan MS/MS mode using a multilevel calibration
graph with deuterated cytokinins as internal standards. The levels of
27 different cytokinin derivatives were measured. The detection limit
was calculated for each compound as 3.3 r/S, where r is the standard
deviation of the response and S the slope of the calibration curve.
Three independent experiments were done. Each sample was injected
at least twice.
Determination of CKX activity
The enzyme preparations were extracted and partially purified using
the method of Chatfield and Armstrong (1986) as modified by
Motyka et al. (2003). The CKX activity was determined by in vitro
assays based on the conversion of [2-3H]isopentenyladenine (prepared by Dr Jan Hanuš) to [3H]adenine. The assay mixture (50 mm3
final volume) included 100 mM TAPS-NaOH buffer containing
75 lM 2,6-dichloroindophenol (pH 8.5), 2 lM substrate ([2-3H]
isopentenyladenine, 7.4 Bq molÿ1) and the enzyme preparation
equivalent to 250 mg tissue FW (corresponding to 0.02–0.26 mg
protein gÿ1 FW). After incubation at 37 8C the reaction was
terminated by the addition of 10 mm3 Na4EDTA (200 mM) and
120 mm3 95% (v/v) ethanol. Separation of the substrate from the
product of the enzyme reaction was achieved by the same HPLC
system as described for tritiated trans-zeatin metabolites according
to Blagoeva et al. (2004). The CKX activity was determined in two
independent experiments. As the data from both experiments showed
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polar transport (Tian and Reed, 2001) or by the modulation
of cell sensitivity towards auxin (Jones et al., 1991).
The data on the effect of light on the metabolism of
cytokinins are rather scarce. The assumption of a positive
effect of light on CK accumulation has been based on the
decreased level of trans-zeatin in phytochrome mutants
(Kraepiel et al., 1995). Diurnal variation of CK levels was
followed in barley (Kurapov et al., 2000) and carrot
(Paasch et al., 1997), but these studies were limited only
to the determination of CK bases and ribosides. The recent
development of analytical methods (especially of mass
spectrometry) has enabled the study of the diurnal variation
of a broad array of CK metabolites.
Diurnal changes in the levels of 27 CK metabolites were
studied in tobacco leaves. The potential contribution of the
cleavage of CK O-glucoconjugates to the fast changes of
the level of physiologically active CKs (CK bases and
ribosides) was estimated via the activity of b-glucosidase
(EC 3.2.1.21). In order to study CK catabolism, the activity
of the main CK-degrading enzyme, cytokinin oxidase/
dehydrogenase (CKX, EC 1.5.99.12) was determined. As
CK levels are also affected by other hormones, the diurnal
variation of auxin and ABA levels was followed.
Cytokinin, auxin, and ABA diurnal rhythms
2879
the same tendencies (but different absolute values), the results of
one representative experiment (mean value of three parallel assays for
each of three replicate protein preparations) are presented.
Determination of b-glucosidase activity
Tobacco leaves (0.2 g FW) were ground in liquid nitrogen and
transferred into 0.4 cm3 ice-cold extraction buffer [25 mM TRIS-Cl,
pH 7.5, 5 mM MgCl2, 1 mM EDTA, and protease Complete EDTAfree cocktail tablet (Roche)]. Samples were centrifuged at 13 000 g
for 15 min (4 8C) and 0.02 cm3 supernatant were added into 0.28 cm3
citrate-phosphate buffer (0.02 M citric acid; 0.06 M NaH2PO4; pH
5.5). The reaction was initiated by 0.1 cm3 20 mM p-nitrophenyl
a-D-glucopyranoside. After 120 min at 30 8C the reaction was
stopped by adding 0.6 cm3 2 M Na2CO3. The released p-nitrophenol
was measured at 405 nm (UNICAM 5625 UV/VIS spectrometer).
Results
Fig. 2. Endogenous levels of CK N-glucosides, CK phosphates, and ciszeatin derivates in tobacco leaves during a 24 h period. Other details as
for Fig. 1.
Cytokinin O-glucosides and activity of b-glucosidase
The level of CK storage forms (O-glucosides) was found
to increase around the dark/light transition. Another peak
of O-glucosides was detected after 7 h of light, preceding
the peak of active CKs as well as that of CK deactivation metabolites. However, this CK O-glucoside peak was
significantly lower than those of the deactivation products.
Minor peaks of CK O-glucosides were detected after 10 h
of light, when the level of active CKs was decreasing, and
2 h after the light/dark transition.
The activity of b-glucosidase, the enzyme that catalyses
the cleavage of glucosyl moieties from O-glucosides releasing, in the case of CK O-glucosides, active CKs,
exhibited the first maximum after 1 h of light, which correlated with the decrease of the peak of CK O-glucosides
found around the dark/light transition (Fig. 3). The major
peak of b-glucosidase activity was found 7 h after the light
was switched on, preceding the midday peak of active CKs.
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Cytokinins
Physiologically active cytokinins, i.e. trans-zeatin, isopentenyladenine, dihydrozeatin, and their corresponding ribosides, exhibited in tobacco leaves a sharp maximum after
9 h of light, i.e. 1 h after the middle of the light period
(Fig. 1). Another, considerably lower, peak was detected
after the transition to darkness and the lowest one was
found at the beginning of the light phase. The content of
cis-zeatin and its riboside exhibited a minor peak coinciding with the maximum of active CKs. Their maximum
followed the second peak of active CKs after the light/
dark transition (with 2 h delay). The content of total CKs
(the sum of physiologically active CKs together with CK
phosphates, cis-zeatin derivatives, N- and O-glucosides)
have an increasing trend during the light period, up to
a maximum 9 h after the light was switched on (i.e. 1 h after
midday). The second half of the light period was characterized by a sharp decrease in CKs. After the light/dark
transition another transient CK peak occurred. During the
course of the dark period, the total CK level gradually
declined. (The levels of individual CKs are given in a table
as supplementary material that can be found at JXB online.)
The concentration of the CK deactivation metabolites
(N-glucosides) increased at the end of the dark period and
remained at an elevated level up to 6 h of light (Fig. 2). The
main maximum of CK N-glucosides coincided with (or
closely followed) the peak of active CKs after the middle
of the light period. Then, a sharp decrease took place.
The second main peak, nearly as large as the first one, was
found after the light was switched off, coinciding with
a minor peak of physiologically active cytokinins. The
level of cis-zeatin derivatives followed a trend similar to
the N-glucosides, with the exception of the second maximum after the light/dark transition, which was, in the case
of cis-zeatin derivatives, delayed and much less profound.
The levels of CK phosphates exhibited a transient peak
around 4 h of light. Their maximum was found after the
middle of the light period.
Fig. 1. Endogenous levels of total cytokinins, physiologically active
cytokinins (trans-zeatin, isopentenyladenine, dihydrozeatin, and the corresponding ribosides) and cis-zeatin (riboside) in tobacco leaves during
a 24 h period. The photoperiod is represented by the black and white box on
the abscissa. Means 6SE of three independent experiments are shown.
2880 Nováková et al.
Cytokinin oxidase/dehydrogenase
A comparison of the diurnal variations in CKX activity and
in the levels of CKs, which are the substrates of this enzyme,
i.e. isopentenyladenine, trans-zeatin, and their corresponding ribosides (but not dihydrozeatin or its derivatives), is
shown in Fig. 4. The main CKX maximum was found 3 h
after the transition from dark to light. The second maximum followed the midday peak of active CKs. The third,
least profound peak of CKX activity accompanied the
minor peak of CKs after the light/dark transition (with
a 1 h delay).
Metabolic conversion of radiolabelled trans-zeatin
Diurnal changes in the levels of endogenous CKs and the
activity of CK metabolic enzymes were also evaluated
following the metabolic conversion of radiolabelled
trans-zeatin (Fig. 5). A high peak of labelled adenine and
adenosine, products of CK breakdown, was found close to
the dark/light transition and a minor one around 9 h of light,
i.e. at the periods preceding the detected maxima of CKX
activity. The peak of non-metabolized trans-zeatin, corresponding to the period of the diminished trans-zeatin
breakdown, preceded the midday peak of active CKs. The
maximum of the non-metabolized labelled trans-zeatin
correlated with the minimum level of the ribosylated and
phosphoribosylated forms.
Fig. 3. Endogenous levels of CK O-glucosides and activity of
b-glucosidase in tobacco leaves during a 24 h period. Other details as
for Fig. 1.
Fig. 5. Metabolism of radiolabelled trans-zeatin in tobacco leaves
during a 24 h period (incubation time 1 h). Other details as for Fig. 1.
Fig. 4. The activity of cytokinin oxidase/dehydrogenase (CKX) and
endogenous levels of cytokinins, which are the substrates of this enzyme
(trans-zeatin, isopentenyladenine, and the corresponding ribosides), in
tobacco leaves during a 24 h period. Other details as for Fig. 1.
Fig. 6. Endogenous levels of indole-3-acetic acid (IAA), abscisic acid
(ABA), and active cytokinins in tobacco leaves during a 24 h period.
Other details as for Fig. 1.
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Indolyl acetic acid and abscisic acid
The midday CK peak coincided with the maximum level
of indolyl acetic acid and was accompanied (or closely
followed) by a minor peak of ABA (Fig. 6). No other
profound peak of indolyl acetic acid was detected during
the diurnal cycle. At the end of the light period, a gradual
increase in ABA level was found. This enhancement was
accelerated after the light was switched off, the maximum
Cytokinin, auxin, and ABA diurnal rhythms
being reached after 3 h of dark. The high level of ABA was
only transient. A low, sharp peak of ABA was also detected
immediately after the light was switched on.
Discussion
who reported a stimulation of ABA biosynthesis at dark,
in order to maintain the stomata in the closed position
during the night to ensure maximal tissue rehydration.
The sharp decrease in ABA concentration, which was
found shortly after the beginning of the light period, seems
to be connected with the depletion of endogenous
ABA and the activation of its catabolism. According
to Tallman (2004), after irradiation ABA levels drop
due to the activation of the xanthophyll cycle (conversion
of ABA precursors to zeaxanthin), isomerization into
physiologically inactive trans-forms, as well as to ABA
degradation by cytochrome P450 mono-oxygenase.
Phytochrome-mediated activation of ABA degradation
was also reported by Kraepiel et al. (1994). The decrease
in ABA concentration in the guard cells and surrounding
apoplast in the morning enables stomatal opening, which
positively affects plant photosynthesis.
Later, at the midday phase, elevated apoplastic ABA
around guard cells, delivered by transpiration, is necessary
for the regulation of the stomata aperture in order to balance
the rate of photosynthesis and plant turgor maintenance
(Tallman, 2004). The minor peak of ABA, which was found
1 h after the middle of the light phase, might be related
to this ‘after-midday’ regulation or, more probably, may
serve to counterbalance the midday peak of CKs, as under
temperate conditions stomatal aperture is regulated by the
CK/ABA ratio (Fusseder et al., 1992).
Fusseder et al. (1992) reported that the increase in
xylem-delivered CKs preceded the increase in leaf conductance. They found the peak of xylem CKs in the morning
and their rapid decrease in the afternoon in desert-grown
almond trees. Beck and Wagner (1994) suggested that the
high concentration of xylem CKs in the morning originated
from the steady rate of CK biosynthesis in the roots and of
their exudation into the xylem sap during the night when
the transpiration rate is low. In the morning, the flow of
xylem sap sharply increases, delivering accumulated CKs
throughout the plant. Due to the increased volume of the
transpiration stream during the day and the steady rate of
CK biosynthesis in roots, CK concentration in the xylem
sap gradually decreases. The importance of the velocity
of the transpiration stream on the translocation of CKs
produced in roots to other parts of plant was recently
demonstrated by Aloni et al. (2005). In our experiments
xylem transport of CKs was not measured. Indirect evidence of the existence of such translocation may be given
by the position of the main maximum of CKX activity that
was detected early in the light period. The other peaks of
CKX activity (after midday and following the light/dark
transition) correlated well with the decrease of endogenous
CKs in leaf blades, indicating that CK breakdown by CKX
is involved in the down-regulation of these endogenous CK
peaks. However, the main maximum of CKX activity,
which appeared early in the light phase, did not coincide
with substantial changes of endogenous CK levels in leaf
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Timing of the main CK peak in tobacco leaves shortly (1
h) after the middle of the light period is in accordance
with the diurnal profile in barley leaves (main peaks of
CK bases and ribosides around 14.00 h; Kurapov et al.,
2000) and slightly differs from that in carrot plants kept
under continuous light (maximum of CK bases and ribosides around 16.00 h; Paasch et al., 1997). As both CKs and
auxin are necessary for the progression of cell division,
this finding of the coincidence of the main CK peak with
the maximum of IAA indicates that cell division may be
initiated at this time. The close connection between CK
increase and growth stimulation was reported in barley
(at the stage of the third leaf), when the CK peak at 14.00 h
was shown to precede the period of maximal growth from
15.00 h to 18.00 h (Kurapov et al., 2000). The minimal
growth was determined before dawn (at 04.00–05.00 h). A
correlation between growth and the hormone level was
also found in the case of auxin, the link between the oscillation in the level of IAA in the rosette leaves and the stem
growth rate in Arabidopsis was reported by Jouve et al.
(1998, 1999).
The results on the midday peak of IAA are in accordance
with the data of Pavlova and Krekule (1984), who reported
a peak of IAA content after the middle of the light phase in
Chenopodium rubrum plants grown under very long day
(20 h). In shorter periods (16 h, 12 h, 8 h), connected with
flower induction, this peak was shifted towards the dark
phase.
It should be kept in mind that these results reflect the
situation in still developing but not the youngest tobacco
leaves, in other tissues and/or species hormones may
exhibit a different diurnal pattern. The effect of developmental stage was observed in barley. Leaves at the stage of
the third leaf had the main CK peak at 14.00 h, while at the
stage of tillering the peak was shifted to 17.00 h (Kurapov
et al., 2000). Another point is that the experiments were
aimed at the determination of the diurnal profile of isoprenoid CKs. Due to the differences in physiological function, diurnal rhythms of isoprenoid CKs are likely to differ
from those of their aromatic counterparts. The peaks of
aromatic CKs m-methoxytopolin and its riboside were
detected at 09.30 h in poplar leaves (Tarkowska et al.,
2003), which corresponded approximately to 3 h after the
light period initiation, i.e. 6 h earlier than for isoprenoid
CKs in tobacco.
The finding of the ABA maximum at the beginning of
the dark phase is in accordance with the survey of diurnal
changes in ABA metabolism summarized by Tallman (2004),
2881
2882 Nováková et al.
Supplementary data
Supplementary data for this paper can be found at JXB
online.
Acknowledgements
The authors wish to acknowledge Eva Kobzová and Marie Korecká
for skilful technical assistance. This work was supported by grants
from the Grant Agency of the Czech Republic, No. 206/03/0369,
and from the Ministry of Education, Youth and Sports, LN
LN00A081.
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To evaluate the role of CK reversible conjugation in
the regulation of their levels, the diurnal rhythm of bglucosidase activity was followed. In the first hours of
the light period, the increase in b-glucosidase activity
created conditions for the cleavage of CK O-glucosides
(one of the CK transport forms) necessary for their further
degradation with CKX. The b-glucosidase increase could,
of course, also result in hydrolysis of other unidentified
compounds, for example, ABA-glucosylester. However, at
this time-point, when the level of free ABA was decreasing
(Tallman, 2004), the function of b-glucosidase activity
stimulation did not seem to be ABA-glucoside deconjugation. The occurrence of b-glucosidase peak in the period
before and after the main peak of CKs indicates that
hydrolysis of CK O-glucosides may be involved in the finetuning of active CK levels at midday, even if this deglucosylation cannot be the only source of the midday peak
of physiologically active CKs. As CK translocation from
the roots, which is the primary site of CK biosynthesis
(Letham, 1963; Aloni et al., 2005), was reported to be
high in the morning, declining rapidly during the day (Beck
and Wagner 1994), CK biosynthesis seems to be a more
probable source of the sharp midday increase of CKs. As
only free ABA was determined, hardly any conclusion may
be drawn about the role of b-glucosidase in the regulation
of free ABA levels at the ‘after-midday’ peak. This will
be the subject of further research. Determination of the
bulk leaf ABA content represents another methodological limitation, as for regulation of stomata aperture ABA
concentration in the guard cells and the surrounding apoplast is crucial, and the bulk ABA concentration includes
the ABA stored in mesophyll chloroplasts as well.
The physiological significance of CK peaks may be
related, apart from the stimulation of cell division in leaves
or the regulation of stomatal aperture, to the generation
of a potential CK signal to other plant parts (e.g. flower
induction, Macháčková et al., 1996) or the induction/
repression of CK responsive genes (Hare and van Staden,
1997). These genes usually display circadian oscillations
in their levels of abundance with maximal expression
during the light period (Thomas et al., 1997). The circadian
clock represents an important adaptation of life to the
conditions on the earth and provides the necessary external
and internal co-ordination of the metabolic processes.
The results here have shown that CKs, auxin, and ABA
levels undergo significant variations in leaves during the
day and night. These data provide, to our knowledge, the
first report of a diurnal rhythm of CK metabolic enzymes
and CK metabolites other than CK bases and ribosides. The
coincidence of the peak of CKs and auxin indicates that
the changes in plant hormone levels have physiological
relevance and correlate with the diurnal changes of plant
growth and development.
Cytokinin, auxin, and ABA diurnal rhythms
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