PHYSIOLOGIA PLANTARUM 101:689-700. 1997
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Copyright ©Physiologia Plantarum 1997
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Regulation of cytokinin content in plant cells
Miroslav Kaminek, Vaclav Motyka and Radomira Vankova
Kaminek, M., Motyka, V. and Vankova, R. 1997. Regulation of cytokinin content in
plant cells. - Physiol. Plant. 101: 689-700.
Cytokinin levels in plant cells are dependent on cytokinin biosynthesis and/or uptake
from extracellular sources, metabolic interconversions, inactivation and degradation.
Cytokinin conversion to compounds differing in polarity seems to be decisive for their
entrapment within the cell and intracellular compartmentation, which affects their
metabolic stability. Increase in cytokinin levels, resulting either from their uptake or
intracellular biosynthesis, may promote further autoinductive accumulation of cytokinins
which may function in the induction of cytokinin-initiated physiological processes.
Accumulated cytokinins are capable of inducing cytokinin oxidase which consequently
decreases cytokinin levels. This seems to be the mechanism of re-estabhshment
and maintenance of cytokinin homeostasis required for further development of
physiological events induced by transient cytokinin accumulation. Auxin may influence
cytokinin levels by down regulation of cytokinin biosynthesis and/or by promotion
of cytokinin degradation. A model of the regulation of cytokinin levels in plant
cells based on these phenomena is presented and its physiological role(s) is discussed.
Key words - Auxin, cytokinin biosynthesis, cytokinin compartmentation, cytokinin
content, cytokinin oxidase, cytokinin uptake, ipt gene.
M. Kaminek (corresponding author, e-mail kaminek@mbox.cesnet.cz), De Montfort
Univ. Norman Borlaug Centre for Plant Science, Inst. of Experimental Botany, The
Academy of Sciences of the Czech Republic, Ke dvoru 15, 166 30 Prague 6, Czech Republic;
V. Motyka and R. Vankova, Inst. of Experimental Botany, The Academy of Sciences
of the Czech Republic, Ke dvoru 15, 166 30 Prague 6, Czech Republic.
n ro uc ion
ticular physiological processes is really govemed by
changes in levels of endogenous cytokinins. It is obvious
The discovery of cytokinins and investigation of their that both application of exogenous cytokinins and maroles
in plants originated from testing the effects of ex- nipulated expression of the ipt gene in transgenic plant
ogenously applied substances promoting cell division in cells represent useful, if very cmde tools for probing the
a number of plant systems. Exogenous application is as- actual role of cytokinins in plants,
sociated with many well known limitations arising from Mechanisms of metabolic regulation of plant hordifferences
in uptake (Laloue et al. 1981, Auer et al. mone levels should meet two basic requirements essen-
1992), metabolism (Kaminek 1992, McGaw and Burch tial for hormonal control of plant development: (1) the
1995) and compartmentation (Jameson 1994) of applied ability to respond to hormonal or other signals and gensubstances
in plant cells which complicate the interpre- erate fast and significant changes in the concentration of
tation of results. Therefore, it has been encouraging to a particular hormone or its concentration ratio with releam
that most of the biological effects described for ex- spect to other plant hormone(s) in particular cells; and
ogenously applied cytokinins are also induced by ele- (2) the capability to maintain hormonal homeostasis at
vated levels of endogenous cytokinins in transgenic certain stage(s) of cell development. Rapid changes in
plant cells expressing the cytokinin biosynthetic ipt gene hormonal levels function in the initiation of major develcoding
for the enzyme A^-isopentenyl pyrophosphate:5'- opmental processes (e.g. organ formation) while horAMP
A'-isopentenyl transferase (see Klee and Romano monal homeostasis is required for further development
1994). These findings indicate that the regulation of par- of initiated events (e.g. organ growth). Operation of
Received 23 April, 1997
Physiol, Plant, 101, 1997 689
these mechanisms is certainly dependent on cell hormonal
status. Most plant cells and tissues are cytokinindependent
when cultured in vitro, suggesting a strong
dependence of their intracellular cytokinin levels on cytokinin
supply from exogenous sources. However, this
does not imply the cytokinin dependency of cells and
tissues of intact plants, which exhibit limited cytokinin
effiux and different 'cytokinin economy' as compared to
isolated ones.
To understand the precise mechanisms controlling cytokinin
levels and action we need to expand our knowledge
of the uptake of different cytokinins and their compartmentation
in plant cells, pathways and regulations of
cytokinin biosynthesis, metabolic interconversions, inactivation
and degradation and sensitivity of plant cells
to so far poorly identified 'active forms' of cytokinins.
The present contribution focuses on how cytokinin uptake,
compartmentation, autoinductive accumulation and
degradation regulate cytokinin levels in plant cells.
Abbreviations - BA3G, BA-3-glucoside; CKOX, cytokinin oxidase;
DZ, dihydrozeatin; DZMP, dihydrozeatin nucleotide;
DZOG, dihydrozeatin O-yS-glucoside; DZR, dihydrozeatin riboside;
DZROG, dihydrozeatin riboside O-y9-glucoside; iP, A'^-(A^isopentenyl)adenine;
iPA, A'*'-(A'-isopentenyl)adenosine; ipt,
gene encoding isopentenyl transferase; IPT, isopentenyl transferase;
NAA, 1-naphthaleneacetic acid; Z, zeatin; Z7G, zeatin-
7-glucoside; ZMP, zeatin nucleotide; ZOG, zeatin O-fi-g\ucoside,
ZR, zeatin riboside; ZROG, zeatin riboside 0-yff-glucoside,
Cytokinin uptake
The differences in cytokinin uptake from extracellular
solutes including xylem and phloem sap (see Letham
1994, Hoad 1995) by different plant cells may be decisive
for long-distance delivery of hormonal signals and
regulation of cytokinin levels in target plant cells. The
existence of such signals and their possible physiological
function have been supported by a number of reported
correlations between changes in the cytokinin
content in xylem sap and the developmental events that
are under cytokinin control, such as leaf senescence of
monocarpic plants (Sitton et al. 1967, Nooden et al.
1990), release of lateral buds from apical dominance in
Phaseolus vulgaris and Pisum sativum (Bangerth 1994,
Li et al. 1995) and bud burst of deciduous trees (e.g.
Tromp and Ovaa 1990). Recently, Nooden et al. (1990)
correlated the changes in cytokinin concentrations in xylem
exudates with the response to pod removal in Glycine
max plants. Depodding at full pod extension, which
delayed leaf senescence, was accompanied by a dramatic
increase of predominant cytokinins, trans-z&atin
riboside (ZR) and dihydrozeatin riboside (DZR). Pod removal
at late pod-filling did not delay senescence and
had little effect on xylem cytokinins.
Concentration of cytokinins occurring in xylem exudates
seems to be physiologically relevant. Application
of cytokinins to derooted seedlings at multiples of their
endogenous concentrations found in xylem sap was effective
for the retention of leaf senescence in oats and
regulation of transpiration in wheat seedlings (Badenoch-Jones
et al. 1996). However, radioactivity from
[^H]ZR or [^H]DZR supplied to Nicotiana tabacum
plants via xylem was not preferentially accumulated in
young leaves as compared to senescing ones, indicating
that cytokinins in upper young leaves may also originate
from other sources including their biosynthesis de novo
(Singh at al. 1992).
Tobacco cells were found permeable to lipophilic cytokinin
bases (A/*^-benzyladenine [BA], M-[A^-isopentenyljadenine
[iP]) and their respective ribosides, which
become entrapped in the cells after their metabolic conversion
to corresponding polar cytokinin nucleotides, A^and
6>-glucosides (Laloue et al. 1981, Laloue and Pethe
1982) (Fig. 1). Such conversion may be responsible for
local accumulation of cytokinins in individual cells as
described for Nicotiana tabacum leaf cells overexpressing
the ipt gene (Estmch et al. 1991). The resulting shoot
formation was restricted to individual cells and no morphological
symptoms indicating an increase in cytokinin
levels were recognised in neighbouring cells.
The primary role of cytokinin uptake in the regulation
of cytokinin levels in cytokinin-dependent cells was
clearly demonstrated by Palmer and Palni (1986). They
found a similar percentage of [^H]ZR uptake by cytokinin-dependent
callus of Glycine max incubated for 20 h
in solutions differing 100-fold in trans-zeaiin (Z) concentration
indicating that the growth response of this
particular tissue to exogenous cytokinins over a broad
range of concentrations corresponds to the cytokinin up-
take.
(Extracellular)/ (Intracellular) [ (Vacuoie)
CK-ribosides
[CK-O-glucosides)
CK-bases:^
CK-3-glucosides?
Fig. 1. Uptake, metabolic interconversions and compartmentation
of cytokinins in plant cells. (1) Adenine phosphoribosyltransferase,
(2) 5'-nucleotidase, (3) adenine kinase, (4) adenosine
nucleosidase, (5) adenine phosphorylase, (6) (9-glucosyltransferase,
(7) 7(?)-glucosyltransferase, (8) yS-glucosidase. Data
compiled from Palmer et al. 1981, Laloue and Pethe 1982, McGaw
et al. 1984, Fusseder and Ziegler 1988, Kaminek 1992 and
Jameson 1994.
690 Physiol. Plant, 101,1997
Cytokinin compartmentation metabolic release of Z from ZOG may be specific and
3 . localised to certain plant tissues.
Similarly to [ H]ZR in tobacco, the [^HJdihydrozeatin In contrast to cytokinin-0-glucosides, which are bio(DZ)
supplied to photoautotrophically growing cell sus- logically active either themselves or after enzymatic hypension
cultures of Chenopodium rubrum was rapidly drolysis, the cytokinin-N-glucosides are biologically intaken
up and metabolised to ['H]DZR, ['HJdihy- active (see McGaw and Burch 1995). The exception
drozeatin O-glucoside (DZOG) and [^HJdihydrozeatin might be (A03-glucosides which were detected as metabriboside
O-glucoside (DZROG). Gentle osmotic dismp- olites of extemally apphed cytokinins (McGaw et al.
tion and fractionation of protoplasts showed that both 1984). A^-benzyladenine-3-glucoside (BA3G) exhibited
cytokinin-0-glucosides were localised within the vacu- cytokinin activity in a root growth suppression bioassay
ole whereas both [ H]DZ and [^H]DZR were localised and was hydrolysed in vivo in tobacco leaves (Faiss et
outside the vacuoie and part of pHJDZR was excreted al. 1996). If the (A03-glucosides also represent a storage
into the medium (Fusseder and Ziegler 1988). In tomato form of cytokinins and are hydrolysed in the same way
crown gall cells ZR was localised by indirect immu- as 0-glucosides to yield active cytokinins, then their lonogold
labeling only in the cytoplasm (Eberle et al. cahsation in the vacuoie should be considered. Compart-
19^^)- mentation of cytokinins resulting from their metabolic
Several indirect data support compartmentation of cy- interconversions to products differing in their mobility
tokinin-0-glucosides separately from yS-glucosidase ac- across the cell membranes is summarised in Fig. 1.
tivity. 0-glucosides were the major metabolites of Z and
DZ accumulated in detached senescing leaves of
Phaseolus vulgaris (Palmer et al. 1981a,b). This is in ^^egulatory effects of cytokinins and auxin on
contrast with their high activity in leaf senescence assays *^ytokinin levels
(Letham et al. 1983). Zeatin 0-^-glucoside (ZOG) and The complexity of hormonal control in plants is disDZOG
were the two major cytokinins in transgenic to- played by interactions among different classes of plant
bacco callus tissues expressing the ipt gene (Redig et al. hormones in the regulation of different developmental
1997) while zeatin riboside O-/?-glucoside (ZROG) ac- and physiological processes. It is becoming increasingly
cumulated to large amounts in another transgenic to- evident that these interactions also involve alterations of
bacco tissue after derepression of the ipt gene by tetracy- one hormone level by another. In addition, the concencline
(Tc) (Motyka et al. 1996). Comparing the incorpo- tration of each hormone, including cytokinins, is under
ration of exogenously applied [^HJadenine into cytoki- control of until now poorly understood metabolic regulanins
in suspension of tobacco BY-2 cells, most of the ra- tions responding to its own levels in plant cells. In case
dioactivity was associated with ZROG beginning 1 h af- of cytokinins these mechanisms may also include the
ter [^HJadenine application (Dobrev and M. Kaminek, ability of certain plant cells to switch on and off cytokiunpublished
results). Such accumulation seems to be nin biosynthesis and/or degradation,
possible if cytokinin-0-glucosides are not exposed to ySglucosidase.
Although it is believed that ^-glucosidases
are ubiquitous, their precise localisation in tobacco cells Effects of cytokinins on their own accumulation
is not known. High metabolic stability of exogenously An interesting view on mechanisms controlling cytokiapphed
[^H]ZOG and [^H]DZOG in Phaseolus leaves nin levels in plant cells was presented by Meins (1989)
and Raphanus seedlings (McGaw and Horgan 1985) in- who stated that the cell's capacity for cytokinin-autonodicates
that either these conjugates are protected against mous growth is associated with elevated cytokinin levan
attack by yS-glucosidase during their translocation to els. A biochemical-switch model for cytokinin habituathe
vacuoie or the activity of this enzyme is very low in tion proposed that the habituated state is maintained by a
corresponding plant tissues. positive feed-back loop in which cytokinins, or related
Two different yS-glucosidases capable of hydrolysing cell division-promoting factors, either induce their own
ZOG were recently found in Zea mays (Brzobohaty et al. biosynthesis or inhibit their own degradation. Such a sit-
1993) and Brassica napus (Falk and Rask 1995). Both uation seems to exist in meristematic cells. It was rewere
preferentially expressed in young tissues undergo- ported that explants of tobacco pith derived from the miing
intensive cell division and differentiation which are totically active region of Nicotiana tabacum stem apex
known to accumulate cytokinins (Dietrich et al. 1995). were cytokinin-autonomous and habituation frequency
While the B. napus yS-glucosidase was reported to be declined rapidly with increasing distance from the apex
specific for ZOG, the maize enzyme uses ZOG and kine- (Turgeon 1982). Accumulation of cytokinins is indeed
tin-3-glucoside as substrate; however, its potential abil- correlated with the onset of cell division, as shown in
ity to hydrolyse other non-cytokinin y9-glucosides has partially synchronised tobacco cell cultures (Nishinari
not been characterised. Unfortunately, kinetic character- and Syono 1986, Redig et al. 1996, Zazimalova et al.
istics, which can help to elucidate the physiological rele- 1996).
vance of these enzymes, have not been determined. Nev- Levels of endogenous isoprenoid cytokinins were inertheless,
these reports represent the first indication that creased in Glycine max (Thomas and Katterman 1986)
Physiol, Plant, 101, 1997 6 9 1
and Nicotiana (Hansen et al. 1987) callus tissues and in
Beta vulgaris cell suspension (Vankova et al. 1991) following
exposition to exogenous urea-type cytokinin
(thidiazuron) and to cytokinins bearing N^ aromatic or
heterocyclic side chains (BA and kinetin) which can be
readily distinguished from and cannot be converted to
isoprenoid cytokinins. The response of plant cells to exogenous
cytokinin is very fast - a significant increase in
total levels of endogenous cytokinins (preferentially ZR,
DZR, zeatin nucleotide [ZMP], dihydrozeatin nucleotide
[DZMP] and iP) in transgenic tobacco callus expressing
ipt was recorded already 6 h after application of BA solution
to the callus surface and corresponding cytokinin-
0-glucosides accumulated during the following 6 h.
However, an increase in endogenous isoprenoid cytokinin
levels was not recorded in non-transformed tissue
during this short-term experiment (Redig et al. 1997).
Following the dynamics of cytokinin excretion from cytokinin-autonomous
and cytokinin-dependent tobacco
cells immobilised on solid support in a column-fiowthrough
system, Vankova et al. (1987) found that the
former excreted a 4- to 5-fold higher amount of Z and
ZR. Moreover, exposure of the cytokinin-dependent immobilised
cells to a pulse of BA after 72 h of cytokinin
starvation caused a 3- to 5-fold increase in the excretion
rate of Z and ZR. The duration of excretion and the
amount of excreted cytokinins indicated that the excreted
cytokinins were at least partially synthesised de
novo. Recent work (see Zhang et al. 1995) has indeed
shown the promotion of ipt gene expression and promotion
of cytokinin biosynthesis in response to BA which
was detected by northem and westem blotting in transformed
tobacco callus.
Accumulation of isoprenoid cytokinins in response to
application of exogenous cytokinin is capable of inducing
habituation only in tissues which habituate readily.
Exposure of cytokinin-dependent callus tissue of Nicotiana
tabacum cv. Wisconsin 38 to exogenous kinetin did
not induce habituation of the tissue for cytokinins. Fresh
weight of tissue transferred at different time intervals
from kinetin-containing to cytokinin-free medium was
linearly proportional to the time of tissue preculture on
kinetin-containing medium (Fig. 2). This indicates that
either the number of 'inducible' cells in the tissue was
too low or that the elevation of cytokinin levels in the
cells remained under the threshold level required for habituation.
Apparently, the effect of exogenous cytokinins
on the accumulation of the endogenous ones is a widespread
phenomenon in plants; however, the induction of
habituation of cells for cytokinins depends on their competence
for habituation as well as on genetic factors
(Mok et al. 1980, Meins 1989).
It should be mentioned that exogenous auxins in contrast
to cytokinins do not induce accumulation of indole-
3-acetic acid (IAA). Application of 2,4-dichlorophenoxyacetic
acid or naphthaleneacetic acid (NAA) to cultured
hypocotyls of Daucus carota had minor effects on
endogenous IAA concentration. Application of
13
.2f
10 20 30
Callus transfer [days]
40
Fig. 2. The dependence of Nicotiana tabacum callus cv. Wisconsin
38 fresh weight yield on the period of tissue culture on kinetin-containing
medium. Tissue was grown on Murashige-Skoog
agar-solidified medium supplemented with NAA (5 |iM) and kinetin
(0.2 |iM) at 26°C in darkness. At time intervals specified in
the graph the tissue was transferred to the same medium (+K —>
+K) or to kinetin free medium (+K -> -K) and harvested after
40 days of total culture.
promoted IAA conjugation and decreased levels
of non-labelled IAA indicated its negative infiuence
on de novo IAA biosynthesis, possibly by activation of a
feedback mechanism for regulation of its own biosynthesis
(Ribnicky et al. 1996).
Effects of cytokinins on cytokinin oxidase activity
Cytokinin oxidase (CKOX), which catalyses A'^ side
chain cleavage of isoprenoid cytokinins, is the only cytokinin-specific
enzyme known to inactivate cytokinins
by their irreversible degradation. CKOXs from different
plant materials vary in their biochemical properties (pH
optima, molecular masses, Michaelis constants, glycosylation
and stimulation of activity in the presence of imidazole-Cu^"^
complexes; see Armstrong 1994 and Hare
and van Staden 1994a). However, they are very conservative
in their substrate specificity. Studies using different
plant materials such as Zea mays kemels (Whitty and
Hall 1974, McGaw and Horgan 1983), Vinca rosea
crown gall (McGaw and Horgan 1983), wheat germ (Laloue
and Fox 1989), Phaseolus calli (Chatfield and Armstrong
1986, Kaminek and Armstrong 1990), Nicotiana
tabacum and Populus x euroamericana callus (Motyka
and Kaminek 1992a, 1994) showed that iP is the most
preferred substrate. A'^ side chain hydroxylation (forma-
692 Physiol, Plant, 101, 1997
tion of Z) reduces the affinity to CKOX from all plant
materials but Vinca rosea crown gall and 0-glucosylation
protects the conjugates against enzyme attack. The
affinity of CKOX to cytokinins is reduced but not removed
by A/'-substitution of glucosyl or alanyl moieties
on the substrate purine ring (McGaw and Horgan 1983).
Cytokinins bearing a saturated N^ side chain (DZ and
DZR) and cytokinin nucleotides as well as cytokinins
bearing aromatic side chains are not substrates of the
CKOX. However, BA was reported to compete slightly
with A^-(A^-isopentenyl)adenosine (iPA) as a substrate
of CKOX from wheat germ (Laloue and Fox 1989).
If CKOX functions in the regulation of cytokinin levels
it should respond to changes of cytokinin concentration
in plant cells. This was first demonstrated by Terrine
and Laloue (1980) who found that metaboUc degradation
of ["^C]iPA in vivo in cell suspensions of Nicotiana
tabacum increased two- to threefold after exposure
to exogenous iPA and BA. However, Palmer and Palni
(1986) reported decreasing formation of degradative metabolites
of ZR with increasing substrate concentration
in cytokinin-dependent Glycine max callus; nevertheless,
this effect was not significant at low ZR concentra-
tions.
The promotion of CKOX activity assayed in vitro in
response to in vivo application of exogenous cytokinins
was reported for a number of plant systems (Tab. 1).
Detailed analysis of Phaseolus vulgaris and P. lunatus
callus tissues showed that the induction of enzyme activity
is (1) relatively rapid, being detectable already 1 h
after cytokinin application, (2) transient, lasting for not
more than 8 h, and (3) significant, having increased up
to threefold (Chatfield and Armstrong 1986, Kaminek
and Armstrong 1990). RNA and protein synthesis
seems to be required for the CKOX induction as indicated
by its inhibition with cordycepin and cycloheximide
(Chatfield and Armstrong 1986). Similar results
were obtained with Nicotiana tabacum callus, where an
increase in CKOX activity was recorded 2 h after application
of 100 \xM Z solution to callus surface, culminating
after 6 h and lasting for 24 h. The attenuation of enzyme
activity was higher than that of total protein content
(Motyka and Kaminek 1990). Increase in CKOX
activity was also recorded in Glycine max callus after
its long-term cultivation on media supplied with four
different cytokinins, both substrates and non-substrates
of CKOX (Motyka and Kaminek 1992b). All tested cytokinins,
including cytokinin-active derivatives of urea,
were effective as inducers of the enzyme. This indicates
that the cytokinin effect on CKOX activity (at least in
the case of non-substrate cytokinins) is not mediated by
a simple induction of the enzyme by its substrates. Actually
the non-substrate cytokinins exhibited very high
inducing activity in Phaseolus vulgaris callus (Chatfield
and Armstrong 1986, Kaminek and Armstrong
1990) not being degraded by the CKOX to inactive
products (see Tab. 1).
Cytokinin oxidase activity is promoted (surprisingly) by
both substrate and non-substrate cytokinins in vivo
It is an apparent paradox that cytokinins, that are not
substrates of CKOX are capable of inducing two very
contradictory processes, i.e. of accumulation of substrate
cytokinins and CKOX activity.
Several mechanisms of promotion of CKOX by nonsubstrate
cytokinins may be considered. (1) Non-substrate
cytokinins, similar to substrate cytokinins, may be
capable of reacting with repressor(s) and derepress
stmctural gene(s) encoding CKOX. (2) The CKOX may
be an allosteric protein which is activated by both substrate
and non-substrate cytokinins. (3) As described
Tab. 1. Effect of exogenously applied cytokinins to callus tissues of Phaseolus vulgaris, P. lunatus and Nicotiana tabacum and expression
of ipt gene on cytokinin oxidase activity. Tissues were enriched in cytokinins either by application of cytokinin solutions to callus surface
(100 loM, 0.1 m^*' or 0.2 ml'^ g~' fresh weight) or by ipt gene expression prior to determination of cytokinin oxidase activity, NS, statistically
not significant increase. References: "Chatfield and Armstrong 1986, "'Kaminek and Armstrong 1990, ''Motyka and Kaminek 1992a,
'Redig et al. 1996, "^Zhang et al. 1995, ^Motyka et al, 1996, ^Eklof et al. 1996.
Cytokinin
itcdtineni/
ipt gene
expression
Adenine
iP
DZ
Z
cis-Z
BA
BA
Thidiazuron
ipt expression
ipt expression
ipt expression
ipt expression
Physiol, Plant, 101, 1997
P. vulgaris
{% of water control)
103"
281"
_
328"
_
—
-
Cytokinin
oxidase activity
P. lunatus
{% of time zero control)
105"
110"
159"
144"
126"
_
177"
—
—
-
—
N. tabacum
{% of time zero control) or
(% of i/jr-non-transformed control)
104'
2or
i6r
150-200'
156'
500'^
150-300'
200-1000^
NS^
693
above (in Effects of cytokinins on their own accumula- fusion of the gene to constitutive, inducible or organ (tistion)
cytokinins that are not substrates of CKOX are ca- sue) specific promoters. As summarized in Tab. 1 propable
of promoting the accumulation of substrate cytoki- motion of CKOX activity in consequence of ipt gene exnins,
which may subsequently induce CKOX. (4) Phe- pression in tobacco tissues was reported by several aunyl-urea-type
cytokinins may promote CKOX activity thors. According to Zhang et al. (1995) the constitutive
by a distinct mechanism. These synthetic cytokinins are expression of the ipt gene in Nicotiana tabacum callus
known to compete with substrate cytokinins for CKOX grown on hormone-free medium resulted in a 2- to 4.7when
assayed in vitro (Chatfield and Armstrong 1986, fold increase in total cytokinins, which included about a
Kaminek and Armstrong 1990, Motyka and Kaminek 4-fold increase in cytokinins that are substrates of cyto-
1992a, Hare and van Staden 1994b). In this way the kinin oxidase (iP, iPA, Z, and ZR), and a 5-fold elevation
urea-type cytokinins may protect endogenous substrate of CKOX activity. A similar correlation between the accytokinins
which can accumulate and subsequently in- cumulation of total and 'substrate' cytokinins and
duce CKOX in vivo. CKOX activity was found in another Nicotiana tabacum
Nevertheless, there are strong indications that CKOX callus transformed with the ipt gene under the control of
activity in plant cells responds to changes in substrate a light-inducible promoter. Analysis of 16 cytokinins
isoprenoid cytokinin levels. CKOX activity closely par- showed that the transformed tissue contained 25-fold
alleled the content of isoprenoid cytokinins during the higher level of total cytokinins; however, only about
early stages of grain development of cereal plants. Tran- 25% of the cytokinin pool in both tissues represented cysient
peaks of ZR, Z and iP which appeared in Zea mays tokinins that are substrates of CKOX. Enzyme preparakemels
during the intensive cell division phase between tions from //?Mransformed tissue exhibited 1.5-fold
6 and 10 days after pollination (Jones et al. 1992, Lur higher CKOX activity as compared with control tissues
and Setter 1993, Dietrich et al. 1995) were accompanied (Redig et al. 1997).
by a corresponding dramatic increase of CKOX activity The sequence of processes leading to induction of
(Dietrich et al. 1995). Moreover, this culmination in CKOX activity was demonstrated by Motyka et al.
CKOX activity was not recorded in aborting kemels (1996) who exploited the advantage of Nicotiana
(Dietrich et al. 1995) which contain low levels of cytoki- tabacum callus transformed with the ipt gene under the
nins (M. Kaminek, unpublished results). Maize inbreds transcriptional control of the tetracycline-dependent 35S
with high cytokinin levels had also high CKOX activity promoter (Gatz et al. 1992). The ipt gene transcript was
(Cheikh and Jones 1994). Similar correlation was found detected by northem analysis in transformed callus alin
developing grains of Triticum aestivum where tran- ready 2 h after tetracycline application. The levels of
sient accumulation of isoprenoid cytokinins 4-11 days major cytokinins, ZR, ZROG and DZR, began to inafter
anthesis was accompanied by the increase in crease after the following 6 h and progressively inCKOX
activity (Kaminek et al. 1994). Further, accumu- creased for 8 days up to 100-fold as compared with nonlation
of a natural non-substrate cytokinin, N^-{meta-hy- transformed wild-type tissue. An increase in CKOX acdroxybenzyl)adenosine,
which was recorded in wheat tivity, as a consequence of elevated cytokinin levels, was
kemels during grain filling when the substrate iso- recorded 16 to 20 h after gene derepression and was inprenoid
cytokinins remained at basal levels, had no ef- creased up to 10-fold after 13 days. The increase in
feet on the CKOX activity (M. Kaminek and A. Gaudi- CKOX activity in cytokinin-overproducing tissue was
nova, unpublished results). Correlation between cytoki- associated with induction of the glycosylated form of the
nin levels and CKOX activity was also found in leaves enzyme, indicating induction of transcription of a speof
wild-type and genie male-sterile plants of Brassica cific gene. A similar increase in CKOX activity was also
napus. The latter contained lower levels of cytokinins found in detached leaves (8-fold after 4 days) and roots
and was less efficient in degradation of [''^C]Z to (4-fold after 3 days) of transformed plants in response to
['"^C]adenine in vivo as well as in CKOX activity as- tetracycline treatment.
sayed in vitro (Shukla and Sawhney 1997). Induction of Overproduction of cytokinins in transgenic plants exCKOX
by its substrates may be involved in control of pressing the ipt gene is often too high to be physiologifluctuation
of cytokinin levels at distinct stages of the cal. Eklof et al. (1996) analysed tobacco plants with
plant cell cycle being induced by transient cytokinin ac- moderately altered phenotypes expressing the ipt gene at
cumulation at the end of the S phase and during mitosis low levels. As compared to wild-type plants the trans(see
Redig et al. 1996). genie ones accumulated preferentially zeatin-7-glucoside
(Z7G), which is less favorable substrate of CKOX
as compared to Z (McGaw and Horgan 1983), and
Regulation of cytokinin oxidase in transgenic plants CKOX resistant ZMP Unfortunately, determination of
expressing the ipt gene 2R and iPA together with the corresponding nucleotides
Transgenic plants expressing the ipt gene represent a does not allow us to relate levels of the cytokinin ribosuitable
tool for studies of the effects of changes in en- sides to CKOX activity. Both the concentration of Z and
dogenous cytokinin levels on CKOX. The level, dura- CKOX activity were similar in leaves of wild-type and
tion and site of the ipt gene expression can be chosen by transformed plants. However, a higher content of cytoki-
6 9 4 Physiol, Plant, 101, 1997
nins in young leaves, as compared to the old ones, corre- creased tolerance to exogenous auxins and auxin translated
with CKOX activity in both types of plants. port inhibitors due to the expression of the ipt gene under
an auxin-inducible promoter (Li et al. 1994).
Auxin applied exogenously to plant tissues had a simRegulation
of cytokinin levels by auxin -^^^ ^^^^^ ^^ ^^^ lowering of cytokinin levels as that proCytokinins
and auxins interact in the control of many duced endogenously in transformed plants expressing
developmental processes in plants such as cell division auxin biosynthetic genes. Hansen et al. (1987) did not
and differentiation, organ formation in cultured tissues, detect any ZR in cytokinin-requiring and cytokinin-auapical
dominance and leaf senescence (see Thimann totrophic tobacco callus lines when cultured on
1992). Their involvement in initiation and regulation of NAA+kinetin-containing medium, however, both lines
cell division and cell elongation underlines the central accumulated ZR and iPA on the same medium lacking
role of both auxins and cytokinins in plant development auxin. A similar lowering of cytokinin content due to the
and responses of plants to environmental stimuli (see exposure of tobacco cells to exogenous NAA was reHobbie
et al. 1994). Using transgenic plants expressing ported by Beinsberger et al. (1991). Application of a 24the
ipt gene and improved plant hormone analysis tech- h pulse of NAA to immobilised cytokinin-dependent toniques
significant progress has been made during the last bacco cells in a column-fiow-through system allowed
decade in understanding the molecular basis of auxin- Vankova et al. (1992) to monitor the dynamics of excrecytokinin
interactions. tion of cytokinins in response to NAA. The levels of preAs
expected the expression of the ipt gene from Agro- dominant cytokinins ZR and iPA decreased 3- and 4bacterium
tumefaciens in plants of several genera in- fold, respectively, during the first 6 h of cell exposure to
eluding Nicotiana, Arabidopsis, Asparagus, Petunia and NAA. Interestingly, the effect of NAA was transient culKalanchoe
resulted in physiological responses known to minating 6 h after NAA application and the rate of cytobe
induced by exogenous cytokinins. These effects in- kinin excretion retumed to the original levels during the
eluded cytokinin-autotrophy of transformed tissues cul- next 18 h when the cells were still exposed to NAA. This
tured in vitro (Beinsberger et al. 1991, McKenzie et al. dynamics of cytokinin excretion corresponds to the
1994), release of axillary buds and formation of shooty above proposed ability of cells to generate fast changes
phenotype (Medford et al. 1989, Smigocki 1991, Van in hormonal levels in response to extracellular signal(s)
Loven et al. 1993), inhibition of root growth and devel- inducing certain developmental process(es) and subseopment
(Medford et al. 1989, Van Loven et al. 1993, quently to maintain hormonal homeostasis essential for
Hewelt et al. 1994) and retardation of leaf senescence progress of initiated events. By following dynamics of
(Smart et al. 1991, Hewelt et al. 1994). An increase in endogenous cytokinins in an auxin-dependent and cytocytokinin
content up to 600-fold was reported in all kinin-autonomous Nicotiana tabacum cell suspension
these plant systems as a consequence of ipt gene expres- culture, Zazimalova et al. (1996) found striking effects
sion. of exogenous auxin on the cytokinin levels. Culture of
Surprisingly, a dramatic increase in cytokinin content the cells in medium containing one-tenth of the optimum
was found in tumors incited on Nicotiana tabacum stems auxin concentration for growth resulted in a 10-fold inby
Agrobacterium tumefaciens plasmids with mutated crease in cytokinin levels per single cell. Moreover, cyauxin-biosynthesis
genes, while mutation in the ipt gene tokinin bases (iP, Z), which are physiologically more achad
httle effect on auxin levels (Akiyoshi et al. 1983). tive than corresponding ribosides, were preferentially
Changes in the auxin/cytokinin ratio due to the expres- accumulated.
sion of T-DNA mutated in different genes affected tu- Auxin seems to regulate cytokinin levels also in intact
mour morphological pattems in the same way as de- plants (Tab. 2). Removal of apical buds as a source of
scribed by Skoog and Miller (1957) for exogenously ap- auxin or complete disbudding induced rapid and signifiplied
auxin and cytokinin. Similar changes in cytokinin cant increase of cytokinin content and effiux in xylem
and auxin content in tobacco crown gall tissues carrying exudates in several plant species. This effect was accominactivated
or deleted auxin and/or cytokinin biosynthe- panied with physiological and morphological responses,
sis T-DNA genes were reported by McGaw et al. (1988) such as reduction of apical dominance and retention of
and Smigocki and Owens (1989). However, levels of leaf senescence, known to be induced by exogenous cyboth
auxin and cytokinins in non-morphogenic carrot tu- tokinins. Detailed studies of Bangerth's group showed
mours induced by Ti plasmids lacking active auxin bio- that cytokinin content (Z-hZR and iP+iPA) in xylem exusynthetic
genes were similar to those found in tumours date of Phaseolus vulgaris and Pisum sativum seedlings
transformed with wild-type Ti plasmids (Ishikawa et al. was increased 25- and 6- to 10-fold, respectively, within
1988). The inhibiting effect of auxin on cytokinin levels 12-16 h after decapitation. The effect of decapitation
may be responsible for the reported lethal effect of ex- was almost completely reversed after restoration of
pression of gene 7, coding for tryptophan-2-monooxyge- auxin supply by application of NAA on decapitated
nase, catalyzing the first step of IAA biosynthesis in As- shoot (Bangerth 1994, Li et al. 1995). The dynamics of
paragus crown gall tissues (Prinsen et al. 1990). On the the response indicated fast delivery of the auxin signal to
other hand, Nicotiana tabacum plants exhibited in- distant sites of cytokinin synthesis or translocation.
695
Physiol, Plant, 101, 1997
Tab. 2. Enhancement of cytokinin content and stimulation of cytokinin-induced physiological processes by reduction of auxin formation or
removal of auxin sources in plant tissues and intact plants.
Way of reduction
of auxin levels
Inactivation of auxin biosynthetic
genes in tobacco crown gall tumors
Inactivation of auxin biosynthetic
gene in tobacco crown gall tumors
Disbudding of tobacco and tomato
plants
Decapitation of bean
plants
Decapitation of
pea plants
Partial auxin deprivation in culture
medium for tobacco cell suspension
Increase in cytokinin
content (fold)
20
13
4-30
25-40
6-10
10
Physiological effect
Shoot formation
Shoot formation
(not in all tumors)
Delay of senescence
Reduction of
apical dominance
Reduction of
apical dominance
Maintenance of high
rate of cell division
Reference
Akiyoshi et al. 1983
McGaw etal. 1988
Colbert and Beever 1981
Bangerth 1994
Guevara et al. 1995
Zazimalova et al, 1996
Interaction of auxin and cytokinins is further compli- maximally at 25 \iM NAA). This contradiction may be
cated by auxin-induced production of ethylene making it due to the performance of the assay at the same pH (6.5)
difficult to separate effects of auxin on cytokinin and while the pH optimum of tobacco CKOX in imidazole
ethylene levels (Yang and Hoffman 1984). Using trans- buffer is very different from that in other buffers (Kagenic
Nicotiana and Arabidopsis plants overproducing minek and Armstrong 1990, Motyka and Kaminek
auxin and expressing an ethylene synthesis-inhibiting 1- 1994). When tested in Tris-HCl buffer activity of both
aminocyclopropane-1-carboxylate deaminase Romano glycosylated and non-glycosylated forms of purified
et al. (1993) uncoupled overproduction of auxin from CKOX increased by about 50% in the presence of 50 |iM
accumulation of ethylene. This allowed them to demon- NAA. However, the action of auxins on CKOX activity
strate that lateral bud growth in Nicotiana and Arabidop- in vivo may be different as indicated by the application
sis and leaf epinasty in Arabidopsis are under control of of solutions of IAA, NAA and indole-3-butyric acid to
the cytokinin/auxin ratio. surface of tobacco callus (20 |JM final concentration).
None of these auxins affected CKOX activity assayed in
vitro (Motyka and Kaminek 1992a).
How does auxin regulate the cytokinin levels?
Down regulation of cytokinin levels by auxin may occur
at the level of inhibition of cytokinin biosynthesis, or
promotion of cytokinin metabolic inactivation by A^-glucosylation
or degradation by CKOX. Palni et al. (1988)
reported that metabolic stability of [^H]Z supplied to tobacco
stem pith is inversely related to NAA concentration
in incubation medium and proposed that the effect
of auxin on cytokinin metabolism is mediated, at least in
part, through CKOX. Zhang et al. (1995) arrived at the
same conclusion when testing the effect of exogenous
NAA on cytokinin levels and CKOX activity in Nicotiana
tabacum callus expressing the ipt gene. In their experiments
addition of NAA to the culture medium decreased
the total cytokinin content by 84% in transformed
and by 67% in non-transformed tissue and promoted
in vivo conversion of Z-type cytokinins to adenine
derivatives. They did not test the effect of NAA on
CKOX activity extracted from treated tissues. However,
the effect of NAA added to the assay solution at relatively
high concentrations (4- to 10-fold higher than that
of the substrate) on the CKOX activity was dependent
on the type of assay buffer. The CKOX activity was increased
in Tris-HCl buffer (by 90% maximally at 50 [iM
NAA) and decreased in imidazole-CuCl2 buffer (by 33%
tokinin levels by infiuencing cytokinin biosynthesis. Investigating
the effect of the strong synthetic auxin, picloram,
on cytokinin levels in crown gall single cell
clones of Phaseolus vulgaris induced by Agrobacterium
tumefaciens Song et al. (1995) detected ipt transcripts in
tumour tissue cultured on hormone-free medium. However,
ipt RNA was undetectable by northem blot hybridisation
in clones grown on picloram-supplemented medium.
In spite of considerable clonal variation in the cytokinin
contents of crown gall single cell clones, auxin
significantly reduced both the cytokinin content and ipt
mRNA accumulation in all three clones tested. Similar
effects of auxin on the reduction of cytokinin, ipt mRNA
and also IPT protein levels in /pf-transgenic tobacco callus
were reported by Zhang et al. (1996). Levels of cytokinins,
ipt RNA and IFF were decreased by auxin within
4 days, before the occurrence of any change in tissue
morphology. Exogenous BA antagonised the effect of
auxin on cytokinin, ipt mRNA and IPT levels although it
did not directly affect ipt gene expression.
It seems unlikely that auxin regulates cytokinin levels
by affecting both CKOX and cytokinin biosynthesis in
vivo. Data conceming auxin-induced inhibition of ipt
gene expression are less controversial, however, ipt does
696 Physio!, Plant, 101, 1997
Fig. 3. Proposed model
illustrating co-operation of
different regulatory
mechanisms in control of
cytokinin levels in plant cells
and their physiological
role(s).
Extracellular
cytokinin
(natural or synthetic)
Increase of
cytokinin
content
Accumulation of
natural cytokinins.
Promotion of
cytokinin
biosynthesis ?
Induction of
physiological
response(s)
Induction of
cytokinin -
oxidase
Cell wall
Development
of induced
events
Steady state
-*• of cytokinin
content
not seem to be the gene coding for cytokinin biosynthesis
in non-transformed plant cells (see McGaw and
Burch 1995). Nevertheless, inhibition of cytokinin accumulation
in normal plant cells by auxin indicates that the
same or similar regulatory mechanism(s) operates in
both non-transformed and /pr-transformed plant cells.
Proposed model of regulation of cytokinin content in
plant cells
Taking into account the metabolic and physiological responses
to changes in cytokinin supply and/or intracellular
accumulation described, we propose a model for the
regulation of cytokinin content in plant cells (Fig. 3).
According to this scheme the increase in content of endogenous
cytokinins due to their supply from extracellular
sources (xylem or phloem sap, exogenous application)
may induce further cytokinin accumulation by activation
of a positive feedback loop. Having little effect
on auxin levels, the accumulation of cytokinins may also
increase the cytokinin/auxin ratio. Under these conditions
accumulated cytokinins may induce in competent
plant cells certain physiological and/or stmctural processes
such as cytokinin-autonomy and shoot formation.
However, accumulated cytokinins also act as substrate
inducers of CKOX and in this way the cytokinin level
may be subsequently reduced. This seems to be a mechanism
of reestablishment and/or maintenance of cytokinin
homeostasis required for further development of
events initiated by transient cytokinin accumulation.
There is also another developmental altemative: Cells
exhibiting strong positive feedback or weak induction of
CKOX may become cytokinin-autonomous and/or meristematic.
If the positive feedback is based on autoinduction
of cytokinin biosynthesis then such cells may become
new sites of cytokinin biosynthesis.
Auxin may affect cytokinin levels by down regulation
of cytokinin biosynthesis and/or by promotion of
CKOX. In this way auxin may not only decrease the cytokinin
levels but also dramatically decrease cytokinin/auxin
ratio and induce certain physiological events,
such as root formation. Cytokinins may reduce the auxin
effect on their own levels, however, their effect on auxin
levels in plant cells seems to be limited. Nevertheless,
the actual operation of these mechanisms in plant cells
may be expected to differ depending on the cell's developmental
reponse to plant hormones.
Acknowledgments - Assistance of Dipl. Ing. Petre Dobrev and
Dr Eva Zazimalova with computer graphics is greatly appreciated.
This work was supported by grants of the Grant Agency of
The Czech Republic No. 206/96/1032 and 204/96/1424 and of
the Volkswagen Stiftung No. 1112016.
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